JP2007527727A - Lactobacillus acidophilus nucleic acid sequence encoding carbohydrate utilization related protein and use thereof - Google Patents

Abstract

The present invention discloses carbohydrate utilization-related transporters and multidrug transporter nucleic acids and polypeptides, fragments or variants thereof. The present invention also includes a carbohydrate utilization-related and multidrug fusion protein, the same antigenic peptide, and the same anti-carbohydrate utilization-related anti-multidrug antibody. The present invention further provides a vector comprising the nucleic acid of the present invention and a cell into which such a vector has been introduced. A method for producing the polypeptide of the present invention and a method for using the same are also disclosed.
[Selection] Figure 1

Description

  The present invention relates to polynucleotides isolated from lactic acid bacteria, ie, Lactobacillus acidophilus, and polypeptides encoded by such polynucleotides, and further to methods of using such polypeptides and microorganisms that express them. .

This application is a U.S. application number filed March 7, 2005. Is the basis of the priority claim. The name of this basic application is “LACTOBACILLUS ACIDOPHILUS NUCLEIC ACID SEQUENC3ES ENCODING CARBOHYDRATE UTILIZATION-RELATED PROTEINS AND USES THEREFORE”, Todd R. Klaenhammer, Eric Altermann, Rodolphe Barrangou, W. Michael Russell, and Tri Duong are named as inventors, the agent registration number is 5051-693, US provisional application number 60/69 filed on March 8, 2004 Claims the right to be based on 5111121. The entire contents of these are incorporated herein.

  Lactobacillus acidophilus is a Gram-positive, rod-like, non-spore-forming, homofermentative bacterium that is a common resident bacterium in the gastrointestinal tract and urogenital tract, the first in the stool of infants Since its isolation from 1900, this “acid-preferred” organism has been found in the intestinal tract of humans, breastfed infants and those who consume foods rich in milk, lactose and dextrins. Traditionally, Lactobacillus acidophilus has been considered to be an intestinal probiotic that has beneficial effects on the gut microbiota among the Lactobacillus species (Klaenhammer and Russell (2000) "Species of the Lactobacillus acidophilus complex," Encyclopedia of Food Microbiology, Volume 2, pp. 1151-1157, Robinson et al. Eds. (Academic Press, San Diego, California) Lactobacillus acidophilus fermentes hexoses such as lactose and more complex oligosaccharides. Produces lactic acid and lowers the pH of its culture environment Acidic environments (eg, food, vagina, and gastrointestinal tract areas) prevent the growth of undesirable bacteria, pathogens, and yeasts Lactobacillus acidophilus Its acid resistance, viability in cultured dairy products, and viability when passing through the stomach and gastrointestinal tract are well known. Tobacillus and other symbiotic bacteria, some of which are considered “biologically preferred” probiotic bacteria, with regard to their effects on human health, especially prevention or treatment of enteric infections and diarrheal diseases, It has been studied in detail for cancer prevention and immune system stimulation.Lactobacillus has also been studied in terms of its effect on the flavor of dairy products, functional and textural properties. Genetic characterization has been described for Lactobacillus species (eg, L. johnsonii, L. rhamnosus) (see, eg, US Pat. Nos. 6,476,209, 6,544,772, US Patent Publications). 20022015997, 2003013882, 20040009490, International Publication No. 2004/0. 1389, the same 2003/084989, see the same 2004/020467).

  In order for bacteria to grow, a special transport system is needed to take in nutrients from the outside environment. Lactic acid bacteria use the following three transport systems to transport molecules into and out of cells. These transport systems are primary transport, secondary transport, and group translocation. In primary transport, chemical (mainly ATP), electrical, or solar energy is used as the driving force for transport. As a primary transport system in lactic acid bacteria, an ATP binding cassette (ABC) transporter is most frequently used. This transport system is a substrate that crosses the membrane to incorporate sugars and compatible solutes, and to discharge products such as drugs or toxins that are undesirable for the cell and cellular components that function outside the cell (cell wall polysaccharides). The transfer involves ATP hydrolysis. In general, ABC transporters are relatively substrate specific, but some are multispecific, such as multidrug transporters.

  In secondary transport systems, an electrochemical gradient is used to supply energy for carbohydrate transfer. This transport system includes a symporter that co-transports two or more solutes, a uniporter that transports a single molecule, and an antiporter that counter-transports two or more solutes. In general, a symporter couples the uphill movement of the substrate to the proton (or ion) downhill movement, the antiporter uses an ion gradient to secrete the product, and the uniporter (Poolman (2002) Antonie van Leeuwenhoek 82: 147-164).

  Population transfer involves a phosphoenolpyruvate (PEP) -dependent phosphotransferase system (PTS), which causes phosphorylation after uptake of carbohydrates or alditols (Poolman (2002) above). . The phosphate group is brought about by the conversion of PEP to pyruvate, and the subsequent phosphorylation involves the energy binding protein, enzyme I, HPr as well as the substrate specific phosphoryl transfer proteins IIA, IIB, IIC. .

  Multidrug transporters are roughly classified into secondary multidrug transporters and ABC transporters. In addition, secondary multidrug transporters are divided into different families. These families include the major facilitator superfamily (MFS), small multidrug resistance family (SMR), resistance-nodulation-cell division family (RND). family), as well as the multidrug and toxic compound extrusion family (MATE) (Putman et al. (2000) Microbiol. Mol. Biol. Reviews 64: 672-693). Secondary multidrug transporters push drugs out of cells using an electrochemical gradient as described herein. ABC-type multidrug transporters excrete drugs from cells using energy obtained from ATP hydrolysis (Putman et al. (2000) above).

Bacteria can metabolize various carbohydrates not only by using various regulatory mechanisms such as catabolite suppression but also by using transport proteins and transport enzymes having different carbohydrate specificities. The isolation and characterization of these proteins enables the development of important probiotic products that have many possible applications. Such products include those that contribute to the promotion of human and / or animal health and those that are related to food production and safety. These proteins can also be used to develop transgenic plants with altered growth and viability.
US Pat. No. 6,476,209 US Pat. No. 6,544,772 US Patent Publication No. 20020159996 US Patent Publication No. 2003013882 US Patent Publication No. 20040009490 International Publication No. 2004/031389 International Publication No. 2003/08489 International Publication No. 2004/020467 Klaenhammer and Russell (2000) Volume 2, pp. 1151-1157 Robinson et al. Eds. (Academic Press) Poolman (2002) Antonie van Leeuwenhoek 82: 147-164 Putman et al. (2000) Microbiol. Mol. Biol. Reviews 64: 672-693

  The present invention provides compositions and methods for modifying microorganisms and plants. The composition of the present invention includes a nucleic acid encoding a carbohydrate utilization-related protein and isolated from Lactobacillus acidophilus, which includes a phosphotransferase system (PTS) protein, ABC, Transporters and proteins involved in sugar transport, degradation and / or synthesis in Lactobacillus acidophilus are included. The composition also includes a nucleic acid isolated from Lactobacillus acidophilus encoding a multidrug transporter.

  Specifically, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence (individually and / or in any combination) represented by an odd number in SEQ ID NOs: 1 to 363, substantially from these nucleotide sequences. An isolated nucleic acid molecule and / or an isolated nucleic acid molecule consisting of these nucleotide sequences, and an amino acid sequence represented by an even number among SEQ ID NOs: 2-364 (alone and / or in any combination) An isolated nucleic acid molecule encoding is provided. In addition, the nucleic acid molecule encoded by the nucleic acid molecule described herein includes an amino acid sequence and / or an amino acid sequence represented by an even number among SEQ ID NOs: 2 to 364 (alone and / or in any combination). Also provided is an isolated and / or recombinant polypeptide consisting of and / or consisting of. Various nucleic acids and polypeptides having a sufficient degree of identity with the nucleotide sequences and amino acid sequences described in the sequence listing are also encompassed by the present invention. Furthermore, fragments of nucleotide sequences and amino acid sequences described in the sequence listing and fragments having a sufficient degree of identity are also encompassed by the present invention. Nucleotide sequences that are complementary to the nucleic acid sequences of the invention or that hybridize to the nucleotide sequences of the invention are also encompassed by the invention.

  The compositions further include vectors for recombinant expression of the nucleic acid molecules described herein, prokaryotic cells, eukaryotic cells, and plant cells, and transgenic microorganisms and transgenic plants having these vectors. Groups are also included. The present invention also includes a method for producing the polypeptide of the present invention recombinantly and a method for using the recombinantly produced polypeptide. Further included are methods and kits for detecting the presence or absence of nucleic acid and / or polypeptide sequences of the present invention in a sample, and antibodies that bind to the polypeptides of the present invention. Also included in the invention are biologically pure cultures of bacteria having the nucleotide or amino acid sequences of the invention. Foods containing such cultures are also included, such as milk, yogurt, curd products, cheese, fermented milk, ice cream, fermented cereal products, milk powder, infant milk, tablets, bacterial suspensions Oral dry supplements, oral liquid supplements and the like.

  The carbohydrate utilization-related molecule and multidrug transporter molecule of the present invention are useful for selection and production of recombinant bacteria, particularly for the production of bacteria with improved fermentation ability. Such bacteria include bacteria with altered ability to synthesize, transport, accumulate and / or utilize various sugars, bacteria with altered flavor and texture, bacteria producing altered sugars, food processing processes and / or Alternatively, bacteria having improved survival under stress conditions such as the gastrointestinal tract of animals may be mentioned, but the present invention is not limited thereto. Multidrug transporter molecules of the present invention include molecules that improve bacterial viability even when contacted with antimicrobial polypeptides such as bacteriocin and other toxins. These carbohydrate utilization-related molecules and multidrug transporter molecules are also useful for modifying plant species. A transgenic plant having one or more of the sequences of the present invention is considered to be economically effective because it exhibits high tolerance to environmental stresses including (but not limited to) phytopathogens, high salt concentrations, and dehydration. Such transgenic plants are also excellent in resistance to food processing processes and storage conditions.

  The present invention provides an isolated nucleic acid selected from the group consisting of: That is, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, A nucleic acid comprising, consisting of and / or consisting essentially of nucleotide sequences shown in any combination including duplication of the same sequence in 57, 359, 361 and / or 363, and / or its complementary sequence , SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251,253, 255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295, 297, 299, 301, 303, 305, 307, 309, 311, 3 3,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and And / or comprises and / or consists essentially of a nucleotide sequence having a sequence identity of at least 90% with a nucleotide sequence shown in any combination that also includes duplication of the same sequence at 363 , Nucleic acids and SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307 , 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357 A nucleic acid comprising, consisting of and / or consisting essentially of a fragment of a nucleotide sequence shown in any combination that also includes duplication of the same sequence in 359, 361 and / or 363 or a complementary sequence fragment thereof 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 2, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or Amino acids shown in any combination including duplication of the same sequence at 364 A nucleic acid encoding a polypeptide comprising a sequence and / or an amino acid sequence encoded by a nucleic acid molecule described herein, or SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 34, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or any amino acid sequence shown in any combination including duplication of the same sequence And / or a nucleic acid encoding a polypeptide comprising an amino acid sequence encoded by a nucleic acid molecule described herein, or a nucleic acid that hybridizes with any of the above under stringent conditions.

  The composition further includes a vector comprising a nucleic acid described herein, a vector further comprising a nucleic acid encoding a heterologous polypeptide, and a cell (bacterial cell, plant cell, eukaryotic cell, etc.) having such a vector Is included. The present invention also includes a method for recombinant production of the polypeptide of the present invention and a method for using the same. Further included are methods and kits for detecting the presence or absence of the nucleic acid or polypeptide sequences of the present invention in a sample, and antibodies that bind to the polypeptides of the present invention.

  The present invention further provides an isolated polypeptide selected from the group consisting of: A) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 56, 358, 360, 362 and / or 364 comprising any amino acid sequence shown in any combination including duplication of identical sequences and / or amino acid sequences encoded by the nucleic acid molecules described herein, A polypeptide consisting essentially of and / or b) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 3 0, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or 364 encoded by any fragment of the amino acid sequence shown in any combination including duplication of the same sequence and / or nucleic acid molecules described herein A polypeptide comprising, consisting of and / or consisting essentially of a fragment of an amino acid sequence, c) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 6 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or amino acid sequences shown in any combination including duplication of identical sequences and / or as described herein A sequence having at least 90% sequence identity with the amino acid sequence encoded by the nucleic acid molecule A polypeptide comprising, consisting of and / or consisting essentially of a nonacid sequence, or d) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 1 3, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339 , 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and / or a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown E) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 35, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235,237,239,241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283, 285, 287, 289, 291, 293, 295, 297, 299, 3 1, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, Provided are polypeptides encoded by the nucleotide sequences shown in any combination of 351, 353, 355, 357, 359, 361 and / or 363.

  The invention also provides polypeptides of the invention that further comprise one or more heteroamino acid sequences, as well as antibodies that selectively bind to the polypeptides described herein.

  Furthermore, the present invention provides a method for producing a polypeptide, comprising culturing the cell of the present invention under conditions where a nucleotide encoding a polypeptide selected from the group consisting of a) to e) below is expressed. a) SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 92, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 A polypeptide comprising an amino acid sequence shown in any combination including duplication of the same sequence in 8, 360, 362 and / or 364 and / or an amino acid sequence encoded by a nucleic acid molecule described herein, b) a sequence Numbers 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50 , 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 , 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 1 46, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 31 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 And / or a polypeptide comprising a fragment of the amino acid sequence shown in any combination including duplication of the same sequence at 364 and / or a fragment of the amino acid sequence encoded by the nucleic acid molecule described herein, c) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 9 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196 , 198,200,202,204,206,208,210,212,214,216,218,220,222,224,226,228,230,232,234,236,238,240,242,244,246 248, 250, 252, 254, 256, 258, 260, 262, 2 64, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and And / or amino acid sequences shown in any combination that also includes duplication of identical sequences at 364 and / or amino acid sequences having at least 90% sequence identity with the amino acid sequences encoded by the nucleic acid molecules described herein Polypeptide, d) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 9, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 20 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305 , 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355 357, 359, 361 and / or 363 A polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence shown in any combination comprising, and e) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 15 161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199,201,203,205,207,209 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259 , 261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291,293,295,297,299,301,303,305,307,309 311 313 315 317 319 321 323 325 327,329,331,333,335,337,339,341,343,345,347,349,351,353,355,357,359,361 and / or encoded in the nucleotide sequence shown in any combination of 363 Polypeptide.

  Further provided is a method for detecting the presence or absence of a polypeptide in a sample. This method comprises contacting a sample with a compound that selectively binds to a polypeptide and examining whether the compound binds to the polypeptide in the sample, wherein the polypeptide comprises the following a) to e: ). a) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 19 , 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241 243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 A polypeptide encoded by the nucleotide sequence shown in any combination of 359, 361 and / or 363, b) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123 , 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 1 67, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 33 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and / or a fragment of the amino acid sequence encoded by the nucleic acid sequence shown in any combination of 363 A polypeptide comprising c) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 3 05,307,309,311,313,315,317,319,321,323,325,327,329,331,333,335,337,339,341,343,345,347,349,351,353, A polypeptide encoded by a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence shown in any combination of 355, 357, 359, 361 and / or 363, d) SEQ ID NOs: 2, 4, 6, 8 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58 , 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 1 0, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 26 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or 364 A polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in any combination of e) and e) SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 , 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 5 2, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 23 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280 , 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330 A polypeptide comprising the amino acid sequence shown in any combination of 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or 364.

  Further provided is a method for detecting the presence or absence of a polypeptide in a sample. This method comprises contacting a compound that selectively binds to a polypeptide with a sample to determine whether the compound binds to the polypeptide of the present invention in the sample, and the compound that binds to the polypeptide Is an antibody. Also provided is a kit comprising a compound for use in the methods of the invention and instructions for use.

  The present invention also provides a method for detecting the presence or absence of the nucleic acid molecule of the present invention and / or a fragment thereof in a sample. This method comprises: a) contacting a sample with a nucleic acid probe or nucleic acid primer that selectively hybridizes with nucleic acid molecules and / or fragments; and b) whether the nucleic acid probe or nucleic acid primer hybridizes with a nucleic acid molecule in the sample. A method comprising examining and thereby detecting the presence or absence of a nucleic acid molecule of the invention and / or a fragment thereof in a sample. Furthermore, the present invention also provides a method for detecting the presence or absence of the nucleic acid molecule of the present invention and / or a fragment thereof, which comprises contacting a sample containing mRNA molecules with a nucleic acid probe. Also provided is a kit comprising a compound that selectively hybridizes to the nucleic acid of the invention and instructions for use.

  Furthermore, 1) a method for modifying the ability of a living organism to transport carbohydrates into and out of cells, 2) a method for modifying the ability of a living organism to accumulate carbohydrates, and 3) using a carbohydrate as an energy source in a living organism. 4) a method for modifying the ability to produce a modified carbohydrate in an organism, 5) a method for modifying the flavor of a food fermented with microorganisms, and 6) a food fermented with microorganisms. 7) A method of modifying the ability of a living organism to survive under food processing and storage conditions, 8) A method of altering the ability of a living organism to survive in the gastrointestinal tract, 9) A drug of a living organism 10) a method for modifying the ability to transport cells into and out of cells, and 10) a method for modifying the ability to produce carbohydrates in an organism, wherein said organism and / or microorganism is subjected to Vectors comprising one or a plastid sequences, and / or, which method comprises introducing one or more nucleotide sequence consisting of the group consisting of the following sequences of a to e. a) SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 19 , 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241 243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,289,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 Nucleotide sequences shown in any combination of 359, 361 and / or 363, b) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 17 3, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and / or a nucleotide sequence comprising a fragment of the nucleotide sequence shown in any combination of 363, c) SEQ ID NO: 1, 3, 5 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 3 15, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and / or A nucleotide sequence having at least 90% sequence identity to the sequences shown in any combination of 363 and encoding a polypeptide that retains activity, d) SEQ ID NOs: 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 04, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 2 0, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or any of 364 A nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in the combination and retaining activity, and e) SEQ ID NOs: 2, 4, 6, 8, 10 , 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362 and / or any combination of 364 Nucleotide sequence encoding a polypeptide comprising the amino acid sequence shown

  The present invention further relates to 1) a Lactobacillus acidophilus strain with an altered ability to transport carbohydrates into and out of cells, compared to wild-type Lactobacillus acidophilus, and 2) compared to wild-type Lactobacillus acidophilus, Lactobacillus acidophilus strain with altered ability to accumulate carbohydrates, 3) Lactobacillus acidophilus strain with altered ability to use carbohydrate as an energy source compared to wild type Lactobacillus acidophilus, 4) Wild Lactobacillus acidophilus strain that provides food modified in flavor by fermentation compared to Lactobacillus acidophilus type 5) Lactobacillus acidophilus that provides food modified in texture by fermentation compared to wild type Lactobacillus acidophilus Acidophilus strain 6) Wild type Lactobacillus Lactobacillus acidophilus strain modified in ability to produce carbohydrates compared to acidophilus, 7) Lactobacillus modified in ability to survive under food processing and storage conditions compared to wild type Lactobacillus acidophilus Acidophilus strain, 8) Provided is a Lactobacillus acidophilus strain that has an altered ability to survive in the gastrointestinal tract compared to wild-type Lactobacillus acidophilus. The modified abilities, flavors and / or textures described above are listed in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 18 , 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332 334, 336, 338, 340, 342, 344, 346, 348 350, 352, 354, 356, 358, 360, 362 and / or 364, and / or 364 are used as a result of the expression of one or more carbohydrate utilization-related polypeptides represented by sequences arbitrarily combined. is there.

  The present invention further provides a Lactobacillus acidophilus strain with altered viability when contacted with an antimicrobial polypeptide or toxin as compared to wild-type Lactobacillus acidophilus. One or more polypeptides are expressed from the multidrug transport polypeptide represented by an even number among SEQ ID NOs: 78 to 88, 92 to 94, 124 to 126, 132, 282 to 288, 308 and / or 312 to 322. This modifies the above capabilities.

The present invention also relates to a DNA construct comprising one or more of the nucleotides of the present invention, and / or a) any one of nucleotide sequences shown in SEQ ID NOs: 1 to 363, and / or any combination thereof, Complementary sequence, b) any of the nucleotide sequences shown in SEQ ID NOs: 1-363, and / or any combination thereof, or a nucleotide sequence having at least 90% sequence identity with their complementary sequence, c) SEQ ID NO: Any one of nucleotide sequences represented by 1 to 363 alone and / or any combination thereof, or a nucleotide sequence comprising a fragment of a complementary sequence thereof, d) comprising an amino acid sequence represented by any one of SEQ ID NOs: 2 to 364 A nucleotide sequence encoding a polypeptide, e) an amino acid sequence represented by any of SEQ ID NOs: 2-364 And a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 90% sequence homology, and f) a nucleotide sequence that hybridizes under stringent conditions with any of the sequences a) to e) above. Also provided are plants, plant cells, and / or plant seeds that have been stably introduced into its genome with one or more of the nucleotide sequences of the present invention selected from the group.
Detailed Description of the Invention

  The present invention relates to a carbohydrate utilization-related molecule and a multidrug transport molecule derived from Lactobacillus acidophilus, and provides the nucleotide sequence and amino acid sequence of the carbohydrate utilization-related molecule and the multidrug transport molecule. Such sequences are useful for the modification of microorganisms, cells and plants with enhanced properties.

  In the present specification, “a”, “an”, and “the” may be plural or singular throughout the present specification and claims. For example, “a” cell can mean either a single cell or multiple cells.

  Also, in this specification, “and / or” means all of one or more of the associated list items as well as no combination when interpreted by an alternative phrase (“or”). Or any possible combination and includes such combinations.

  A “carbohydrate utilization-related” molecule or gene is a lactobacillus that encodes a protein involved in the utilization of carbohydrate molecules, such as, but not limited to, synthesis, transport, and degradation of carbohydrates. It means a novel sequence derived from Acidophilus. By “multidrug transporter” molecule is meant an antimicrobial polypeptide such as bacteriocin or a molecule involved in the transport of other drugs and toxins. See Table 1 for specific examples of carbohydrate utilization related molecules and multidrug transport molecules of the present invention. These full-length gene sequences are described as “carbohydrate utilization-related sequences” or “multi-drug transporter sequences”, and they are similar to carbohydrate utilization-related genes or multi-drug transporter genes, respectively. Indicates. The present invention further provides fragments or variants of these carbohydrate utilization related sequences or multidrug transporter sequences, which can also be used in practicing the methods of the invention.

  “Carbohydrate” means an organic compound having carbon, hydrogen, and oxygen, usually in a ratio of 1: 2: 1. Carbohydrates include but are not limited to sugar, starch, cellulose and gum. As used herein, the terms “gene” and “recombinant gene” refer to a nucleic acid having an open reading frame, particularly a nucleic acid encoding a carbohydrate utilization-related protein or a multidrug transporter protein. To do. The isolated nucleic acid in the present invention includes a nucleotide sequence encoding a carbohydrate utilization-related protein or a multidrug transporter protein, a nucleic acid sequence encoding an amino acid sequence represented by an even number among SEQ ID NOs: 2 to 364, SEQ ID NO: Nucleic acid sequences indicated by odd numbers from 1 to 363, and variants and fragments thereof are included. The present invention also includes an antisense nucleic acid described below.

  Furthermore, the present invention includes isolated polypeptides and proteins having carbohydrate utilization-related activity or multidrug transport activity, variants or fragments thereof, and methods for producing these polypeptides. In the context of the present invention, the terms “protein” and “polypeptide” are used interchangeably. The polypeptide of the present invention has carbohydrate utilization-related protein activity or multidrug transport activity. Glucose utilization related protein activity or multidrug transport activity refers to biological or functional activity measured in vivo or in vitro by standard assays. Such activities include the ability to synthesize carbohydrates, the ability to transport carbohydrates into and out of cells, the ability to resolve carbohydrates, the ability to regulate intracellular sugar concentrations, the ability to bind carbohydrates, and the drugs or toxins to and from cells. But not limited to these activities.

  Various bacterial transporter structures are well known in the art. The transporter's ATP binding cassette (ABC) superfamily (PFAM deposit number: PF00005) is composed of proteins with four core domains (Higgins et al. (1986) Nature 323: 448-450; Hyde et al. (1990) Nature 346: 362-365; Higgins (2001) Res. Microbiol.152: 205-210) Such proteins typically have a transmembrane domain (PFAM accession number) with 6 transmembrane α-helices each. : PF00664) and two ATP-binding domains with core amino acids that define the transporter (Higgins (2001) above), and other conserved ones such as Walker A motif and Walker B motif. (Walker et al. (1982) EMBO J. 1: 945-951; PROSITE science reference number: PDOC00185).

  The ABC transporter protein of the present invention includes SEQ ID NOs: 40, 42, 44, 48, 52, 54, 56, 58, 62, 64, 66, 68, 70, 72, 74, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 144, 146, 148, 152, 154, 160, 236, 262, 274, 278, 280, 294, 296, 298, 300, 302, 306, 338, 340, and 360 are included. SEQ ID NOs: 126 and 144 are members of the ABC transporter transmembrane region family (PFAM accession number PF00664).

  The TOBE domain (Transport Association OB) (PFAM Accession No. PF03459) always appears as a dimer (Coonin et al. (2000) Adv. Protein Chem. 54 :) because the C-terminal chains of each domain are provided by partners. 245-75). The TOBE domain is probably involved in the recognition of small ligands such as molybdenum and sulfate. The TOBE domain is found immediately after the ATPase domain in the ABC transporter. The TOBE domain protein of the present invention includes the TOBE domain in SEQ ID NO: 110.

  Secondary transport system proteins include the galactoside-pentose-hexuronide group of translocators (Poolman et al. (1996) Mol. Microbiol. 19: 911-922). These proteins are usually composed of a hydrophobic domain with 12 transmembrane domains and a carboxy-terminal enzyme IIA domain (Poolman et al. (1989) J. Bacteriol. 171: 244-253).

  The phosphotransferase system (PTS) catalyzes phosphorylation during the transfer of sugar substrates across the cell membrane. The mechanism is the transfer of phosphoryl group from phosphoenolpyruvate (PEP) via enzyme I (EI) (PROSITE reference number: PDOC00527) of PTS through enzyme II (EII) (PROSITE reference number: PDOC00528; PDOC00795). Involved, and then it is transferred to the phosphocarrier protein (HPr) (PROSITE reference number: PDOC00318) (PFAM accession number PF00381). The HPr protein has two conserved phosphorylation sites, one of which is an amino-terminal histidine residue that is phosphorylated by enzyme I. The other is a serine residue on the carboxy-terminal side of the protein and is phosphorylated by an ATP-dependent protein kinase (de Vos (1996) Antonie van Leeuwenhoek 70: 223-242). SEQ ID NO: 178 is a member of the PTSHPr component phosphorylation site family (PFAM accession number PF00381).

  The sugar-specific permease (enzyme II) of PTS consists of at least three structurally different domains (IIA, IIB and IIC), which domains may exist fused to a single polypeptide chain and interact with each other. May be present as two or three chains having The IIA domain contains a first permease-specific phosphorylation site, histidine that is phosphorylated by phospho-HPr. The second domain (IIB) is phosphorylated on the basis of permeability at cysteinyl or histidyl residues by phospho-IIA. Finally, the phosphoryl group is transferred from the IIB domain to the sugar substrate in a process catalyzed by the IIC domain. This process is involved in transmembrane transport of sugars. The phosphoenolpyruvate-dependent phosphotransferase system (PTS) EIIA1 family (PFAM accession number PF00358) protein in the present invention includes SEQ ID NOs: 6, 12, 14, 34, 102, 104, 174, 176, 268, 290. Illustrated. Examples of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) EIIA2 family (PFAM accession number PF00359) protein in the present invention include the protein of SEQ ID NO: 36. SEQ ID NO: 36, a fructose specific IIB subunit (PFAM accession number PF02379) is also a member of the PTS system. Examples of the phosphotransferase system (PTS) EIIB family (PFAM accession number PF00367) protein in the present invention include SEQ ID NOs: 12, 14, 32, 34, 102, 104, 268, and 290. Examples of the phosphotransferase EIIC family (PFAM accession number PF02378) protein in the present invention include SEQ ID NOs: 12, 14, 16, 28, 30, 32, 34, 36, 102, 104, 174, 268, 270, 272, 290. Is exemplified.

  The lactose / cellobiose specific family is one of four groups that differ structurally and functionally. The IIAPTS enzyme (PFAM accession number PF02255) usually functions as a homotrimer and is stabilized by a metal ion located in the center. Examples of the PTS system and lactose / cellobiose-specific IIA subunit family protein of the present invention include the protein of SEQ ID NO: 4. The lactose / cellobiose-specific IIB subunit family (PFAM accession number PF02302) is a cytoplasmic enzyme. IIB cellobiose fold shows a structure similar to mammalian tyrosine phosphatase. SEQ ID NO: 170 is exemplified as the lactose / cellobiose-specific IIB subunit protein of the PTS system in the present invention.

  The mannose family is unique in several respects among the PTS permease family. The mannose family is the only family in the PTS family whose members have IID proteins, the only family in which such IIB components are phosphorylated at histidyl residues rather than cysteinyl residues, and Permease members are members that have broad specificity for a number of saccharides, rather than being specific for only one or several saccharides. SEQ ID NOs: 20 and 264 are members of the PTS-based fructose IIA component family (PFAM accession number PF03610). SEQ ID NO: 168 is a member of the IID component of the PTS mannose / fructose / sorbose family (PFAM accession number PF03613). SEQ ID NO: 264 is a member of the PTS-based sorbose subfamily IIB component (PFAM accession number PF03830). SEQ ID NO: 166 is a member of the PTS-based sorbose specific iic component family (PFAM accession number PF03609).

  Various enzymes that catalyze the transfer of the phosphoryl group of phosphoenolpyruvate (PEP) through the phospho-histidine intermediate have been shown to be structurally related (Reizer et al. (1993) Protein Sci. 2: 506-21). All of these enzymes have a common catalytic mechanism that binds to PEP and transfers the phosphoryl group of PEP to a histidine residue. The sequence near the histidine residue is highly conserved. The TIM barrel domain (PFAM accession number PF02896), which is a PEP-utilizing enzyme, is the N-terminal and PEP-utilizing enzyme mobile domain (PFAM accession number PF00391). It is frequently seen that it associates with a domain (INTERPRO: IPR002192). The mobile domain, an enzyme that utilizes PEP, is a “swiveling” β / β / α domain, and in all proteins known to have such β / β / α domains, this domain has mobility. (Cosenza et al. (2002) J. Mol. Biol. 318: 1417-32). The β / β / α domain is frequently associated with the PEP / pyruvate binding domain (INTERPRO: IPR002192), which is a pyruvate phosphate dikinase at its N-terminus. The TIM barrel domain protein that is a PEP-utilizing enzyme in the present invention includes SEQ ID NO: 180. The PEP-utilizing enzyme mobile domain protein of the present invention includes SEQ ID NOs: 180 and 258. The N-terminal family (PFAM accession number PF05524) protein that is a PEP-utilizing enzyme in the present invention includes SEQ ID NO: 180.

  Members of the major facilitator superfamily of multidrug transporters (MFS) have 12 or 14 transmembrane segments. Members of the small multidrug resistance family (SMR) of multidrug transporters are thought to form four tightly packed antiparallel helix bundles. This small multi-drug resistant family member excretes a wide range of toxic compounds from the cell, conferring resistance. Members of the resistance nodulation-cell division family (RND) have one N-terminal transmembrane segment and a C-terminal periplasma large domain (Putman et al. (2000) Microbiol. Mol. Biol. Reviews 64: 672-693). Conserved motifs in each of the above types of multidrug transporters, as well as multidrug transporters belonging to the MFS, SMR, and RND families, as well as specific proteins (with accession numbers) derived from various bacteria It has been described in the literature (Putman et al. (2000) above). Multidrug transporter proteins of the present invention include SEQ ID NOs: 78, 80, 82, 84, 86, 88, 92, 94, 282, 284, 286, 288 and 322.

  The sugar (and other) transporter family (PFAM accession number PF00083) belongs to the main facilitator superfamily. The MFS transporter is a single polypeptide secondary carrier that can transport only small solutes in response to a chemical osmotic ion gradient. All currently recognized MFS permeases have two six-transmembrane segment (TMS) units in a single-chain polypeptide, but three of the 17 MFS families have two more TMS has been found (Paulson et al. (1996) Microbiol. Rev. 60: 575-608). Also, MFS-specific motifs that are well conserved between TMS2 and TMS3, and motifs between MS8 and TMS9 that are related to these motifs but have a low degree of conservation (Henderson and Maiden (1990) Philos. Trans. R Soc. Lond. B. Biol. Sci. 326: 391-410) is found to be a feature in virtually all proteins of more than 300 identified MFS proteins. Sugar (and other) transporter proteins of the invention include SEQ ID NOs: 80 and 282.

  The bacterial binding protein-dependent transport system imparts energy to the periplasma substrate binding protein, one or two homologous and integral integral membrane proteins (PFAM accession number PF00528), as well as the active transport system 1 Alternatively, it is a multicomponent system generally composed of two superficial membrane ATP binding proteins. The key transmembrane proteins described above translocate the substrate across the membrane. Such intramembrane proteins have been shown to have a conserved region located approximately 80-100 residues from the C-terminus (Dassa and Hofnung (1985) EMBO J. 4: 2287-93; Saurin et al. al. (1994) Mol. Microbiol. 12: 993-1004). This conserved region appears to be located in the cytoplasmic loop between the two transmembrane domains (Pearce et al. (1992) Mol. Microbiol. 6: 47-57). These proteins are classified into seven families and are named araH, cysTW, fecCD, hisMQ, libHM, malFG and oppBC, respectively. The membrane protein components of the binding protein-dependent transport system in the present invention include the proteins of SEQ ID NOs: 42, 44, 62, 64, 112, 114, and 294. The branched chain amino acid transport system / permease component family (PFAM Accession No. PF02653) is a large family that mainly includes high affinity branched chain amino acid transporter proteins. Galactose transport permease proteins and ribose transport proteins are also included in this family. Branched chain amino acid transport system / permease component proteins of the present invention include the proteins of SEQ ID NOs: 54, 72 and 74.

  SEQ ID NO: 184 is a member of the HPr serine kinase N-terminal family (PFAM accession number PF02603) and also a member of the HPr serine kinase C-terminal family (PFAM accession number PF07475). The N-terminal family represents the N-terminal region of Hpr serine / threonine kinase PtsK. The C-terminal family represents the C-terminal kinase domain of Hpr serine / threonine kinase PtsK. This kinase is a sensor in the multicomponent phosphate relay system that controls carbon catabolite repression in bacteria (Marquez et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99: 3458-63). This kinase is unique in that it recognizes the tertiary structure of its target and is a member of a novel family that is unrelated to any of the protein kinases reported so far. X-ray analysis of a full-length crystalline enzyme derived from Staphylococcus xylosus at a resolution of 1.95A revealed that the enzyme consists of two distinct domains. These two domains are assembled as a hexameric structure similar to a three-blade propeller. Each of the wings is formed of three N-terminal domains, with a compact central hub associated with a C-terminal kinase domain (Reizer et al. (1998) Mol. Microbiol. 27: 1157-69).

  The periplasma binding protein and sugar binding domain of the LacI family (PFAM accession number PF00532) include periplasma binding protein and LacI family transcriptional regulators. Periplasma binding proteins are the major receptors for the chemotaxis and transport of many glycosolutes. The LacI protein family is a family of transcriptional regulators related to the lac repressor. In this case, the sugar binding domain generally binds to a sugar that alters the DNA binding activity of the repressor domain (lacI). In the present invention, the periplasma binding protein and the sugar binding domain of the LacI family protein include SEQ ID NOs: 38 and 98.

  High affinity transport systems in bacteria are involved in active transport of solutes across the cytoplasmic membrane. Protein components of such transport systems include one or two transmembrane protein components, one or two membrane-associated ATP binding proteins, and a high affinity periplasmic solute binding protein (PFAM accession number PF01547). In Gram-positive bacteria that are covered by a single layer and do not have a periplasmic region, the corresponding protein binds to the membrane via an N-terminal lipid anchor. These homologous proteins themselves do not play an important role in the transport process, but probably trigger or initiate translocation of solutes across the membrane by binding to external sites of intramembrane proteins essential to the efflux system Functioning as a receptor. Also, at least some solute binding proteins are functioning in the initiation of sensory transduction pathways. Bacterial extracellular solute binding proteins of the present invention include the proteins of SEQ ID NOs: 40, 66, 116, 262, 274 and 296.

  The sugar transport protein family (PFAM accession number PF06800) is a family of sugar transporters about 300 residues long in bacteria. Its members include glucose uptake proteins (Fiegler et al. (1999) J. Bacteriol. 181: 4929-36), ribose transport proteins, and presumed and hypothesized to be involved in sugar transport across bacterial membranes. Several membrane proteins above are included. The sugar transport protein of the present invention includes the protein of SEQ ID NO: 234.

  MIP (major endogenous protein) family protein (PFAM Accession No. PF00230) is composed of (1) specific water transport by aquaporins and (2) transport of neutral small solutes such as glycerol by glycerol promoters (Froger et al. (1998) Protein Sci. 7: 1458-68). MIP family proteins are thought to have six transmembrane (6 TM) domains. Sequence analysis suggests that such proteins may have arisen by tandem intragenic replication in ancestral proteins with three transmembrane domains (Wistow et al. (1991) Trends Biochem. Sci. 16: 170- 1).

  Common transport proteins of the present invention include the proteins of SEQ ID NOs: 76, 90, 96 and 194.

  Nucleic acid and protein compositions encompassed by the present invention are isolated or substantially purified. “Isolated” or “substantially purified” refers to a nucleic acid molecule or protein molecule, or biologically active fragment or variant thereof, that contains components normally found in the nucleic acid or protein in its natural state. Means substantially or essentially not contained. Such components include other cellular materials, culture media associated with recombinant production, and / or various chemicals used to chemically synthesize proteins or nucleic acids. The “isolated” nucleic acid in the present invention preferably does not include a nucleic acid sequence adjacent to the target nucleic acid in the genomic DNA of the organism from which the nucleic acid was obtained (eg, present at the 5 ′ or 3 ′ end). Coding sequence). However, the molecule may further contain bases or moieties that do not impair the basic properties of the composition. For example, in many embodiments, an isolated nucleic acid has a nucleic acid sequence that is less than 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb as commonly found in the genomic DNA of the cell from which it was obtained. Similarly, in substantially purified proteins, contaminating or non-carbohydrate utilization related proteins are less than about 30%, 20%, 10%, 5%, or 1% (dry weight). If the protein is produced by recombinant methods, the culture medium is preferably less than 30%, 20%, 10%, or 5% of the amount of the protein preparation, and if the protein is produced chemically, Preferably, the chemical precursor or non-carbohydrate utilization related chemical is less than 30%, 20%, 10%, or 5% (dry weight) of the protein preparation.

  The compositions and methods of the present invention can be used to modulate the function of carbohydrate utilization-related molecules or multidrug transporter molecules of Lactobacillus acidophilus. “Modulate”, “alter”, or “modify” means up-regulation or down-regulation of a target biological activity. The proteins of the present invention are useful for modifying the biological activity of lactic acid bacteria, and are also useful for modifying the nutritional properties or health promoting properties of food fermented by lactic acid bacteria. The nucleotide molecules of the present invention are useful for regulating the expression of carbohydrate utilization related proteins or multidrug transporter proteins expressed by lactic acid bacteria. Also included are up-regulation and down-regulation of expression by the nucleic acids of the invention. Upregulation can be achieved, for example, by providing multiple gene copies, by regulating expression by modification of regulatory elements, by promoting transcriptional or translational mechanisms, or by other methods. Down-regulation can be performed, for example, by using a known antisense method and gene silencing method.

 "Lactic acid bacteria" means the following bacteria: Aerococcus, Carnobacterium, Enterococcus, Lactococcus, Lactobacillus, Leuconostoc ), Oenococcus, Pediococcus, Streptococcus, Melissococcus, Alloiococcus, Dolosigranulum, Lactostraoc, Lactosphaoc Vagococcus and Weissella (Holzapfel et al. (2001) Am. J. Clin. Nutr. 73: 365S-373S; Bergey's Manual of Systematic Bacteriology, Vol. 2 (Williams and Wilkins, Baltimore; (1986) )) genus selected from pp. 1075-1079).

  The polypeptides of the present invention, or microbes that express these polypeptides, are useful as nutritional additives or supplements, and are also useful as additives in dairy products and fermentation processes. The nucleic acid sequences, encoded polypeptides, and microorganisms expressing them are useful for the production of milk-derived products such as cheese, yogurt, fermented dairy products, sour milk, butter milk and the like. The microorganism that expresses the polypeptide of the present invention may be a probiotic organism. “Probiotic” means a living microorganism that has survived in the gastrointestinal tract for generations and has a health effect on the subject. “Subject” refers to an organism that has come into contact with a microorganism that expresses the protein of the present invention. Subject may also refer to humans and other animals.

  In addition to the nucleotide sequence of carbohydrate utilization-related transporters and multidrug transporters and fragments and variants thereof disclosed herein, the nucleic acid of the present invention includes the carbohydrate utilization-related transduction disclosed herein. Also encompassed are homologous nucleic acid sequences identified and isolated from other organisms or cells by hybridizing to complete or partial sequences obtained from porters and multidrug transporters.

(Fragments and variants)
The present invention provides isolated nucleic acids comprising nucleotide sequences encoding carbohydrate utilization related proteins and multidrug transporter proteins, and carbohydrate utilization related proteins and multidrug transporter proteins encoded by such nucleic acids. “Sugar utilization-related protein” means a protein having an amino acid sequence represented by an even number among SEQ ID NOs: 2 to 364. Also provided are fragments and variants of such nucleotide sequences, and the proteins encoded by them. A “fragment” of a nucleotide sequence or protein means a portion of a nucleotide sequence or amino acid sequence.

  The nucleic acid fragments disclosed herein can be used as hybridization probes for identifying nucleic acids encoding carbohydrate utilization-related proteins and nucleic acids encoding multidrug transporters. It can also be used as a primer for amplification techniques (eg, polymerase chain reaction (PCR)) or for mutating carbohydrate utilization-related nucleic acids or multidrug transporter nucleic acids. The nucleic acid fragments of the present invention can be bound to a physical substrate to form so-called macroarrays or microarrays (eg, US Pat. No. 5,838,732; US Pat. No. 5,861,242; WO 89/10977; WO 89/11548). WO 93/17126; see US Pat. No. 6,309,823). Such arrays or “chips” of nucleic acids can be used for gene expression studies or for identification of nucleic acid molecules with sufficient identity to the target sequence.

  The present invention further provides a nucleic acid array or chip as a molecular probe precisely arranged or arranged on a solid support, ie a large number of nucleic acids (eg DNA). Such arrays or chips are currently of interest because of their very small size and high performance in the number of analyses, which can be used to sequence genes, study mutations in genes, and / or Analysis of gene expression becomes possible.

  The function of the nucleic acid array / chip is based on molecular probes, mainly oligonucleotides, and is generally attached to a carrier having a size of several centimeters square or more as required. When performing the analysis, the carrier in the DNA array / chip or the like is coated with a DNA probe (for example, an oligonucleotide) placed at a predetermined position on the carrier. A sample containing a target nucleic acid and / or a fragment thereof (for example, DNA, RNA, cDNA) to be labeled in advance and subjected to analysis is brought into contact with a DNA array / chip, and a double strand is formed by hybridization. The chip surface is analyzed after the washing step and the location of hybridization is determined by the signal emitted from the labeled target. By processing the results of the hybridization fingerprint with a computer, it becomes possible to retrieve information such as gene expression, presence or absence of specific fragments in the sample, determination of sequences, and / or identification of mutations.

  In one embodiment of the present invention, hybridization occurring between the nucleic acid of the present invention used as a probe deposited or synthesized in situ on a DNA chip / array and the target nucleic acid may be performed by fluorescence, radiation, electrical detection, etc. It can be examined by a technique known in the technical field.

  In another embodiment, the nucleotide sequence of the present invention can be used as a DNA array / chip to perform expression analysis of the Lactobacillus acidophilus gene. This analysis is based on a DNA array / chip having probes thereon that select according to its specificity characterizing a given gene or nucleotide sequence. The target sequence to be analyzed is labeled before being hybridized on the chip. After washing, the labeled complex is detected and quantified by performing hybridization at least twice. Differential analysis of RNA derived from a sample is possible from comparative analysis of signal intensity when using the same probe for different samples and / or using different probes for the same sample.

  In yet another embodiment, an array / chip having a nucleotide sequence of the invention can include nucleotide sequences that are specific to other microorganisms, thereby allowing continuous testing and the presence or absence of microorganisms in the sample. Can be quickly identified.

  In yet another embodiment, the DNA array / chip principle can be used to create a protein assay / chip. On such a protein assay / chip, the support is coated with the polypeptides and / or antibodies (or arrays thereof) of the invention instead of nucleic acids. For example, with such a protein array / chip, it is possible to analyze the biomolecular interaction induced by the target being captured with affinity on a support coated with protein or the like by surface plasma resonance (SPR) It becomes. The polypeptide or antibody of the present invention capable of specifically binding an antibody or polypeptide derived from a sample to be analyzed is a protein array / chip for detecting and / or identifying a protein and / or peptide in a sample. Can be used.

  Accordingly, the present invention provides microarrays or microchips having the various nucleic acids of the invention in any combination including repeats, as well as microarrays having the various polypeptides of the invention in any combination including repeats. Also provided are microarrays having antibodies that specifically react with the various polypeptides of the invention in any combination, including repeats.

  “Nucleic acid” means an analog of the above DNA or RNA prepared using DNA molecules (eg, cDNA or genomic DNA) and RNA molecules (eg, mRNA) and nucleotide analogs. The nucleic acid may be either single-stranded or double-stranded, but is usually double-stranded DNA. Nucleotide fragments encoding carbohydrate utilization-related proteins or multidrug transporter proteins encode biologically active protein fragments or be used as hybridization probes or PCR primers as described herein Can do. Biologically active fragments of the polypeptides disclosed herein isolate a portion of one of the nucleotide sequences of the invention and express the coding portion of the protein (eg, in vitro recombination). Expression) and can be prepared by assessing the activity of the encoded protein portion. Fragments of nucleotides encoding carbohydrate utilization related proteins or multidrug transporter proteins have at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, consecutive nucleotides. 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2200, or 2500, including between 5 and 2500, any number not specifically described here, or Carbohydrate utilization related nucleotide sequences or multidrug trans as described herein Includes the number of to nucleotide outstanding during the full length of the over terpolymers nucleotide sequence (e.g. in SEQ ID NO: 1 up to 432, up to 369 in SEQ ID NO: 3).

  Amino acid sequence fragments include polypeptide fragments suitable for use as immunogens to produce anti-carbohydrate utilization related antibodies or anti-multidrug transporter antibodies. The fragment is sufficiently identical to the amino acid sequence of the carbohydrate utilization-related protein or multidrug transporter protein or partial length protein of the present invention, or has an amino acid sequence derived from such a sequence, and the carbohydrate utilization-related protein or Peptides having at least one of the activities of the multidrug transporter protein are included. However, the amino acids contained in the fragments are fewer than the amino acids in the full-length proteins described herein. Usually, the biologically active portion includes a domain or motif having at least one activity of a carbohydrate utilization-related protein or a multidrug transporter protein. The biologically active portion of the carbohydrate utilization-related protein or multidrug transporter protein has a length of, for example, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500. 550, 600, 650 polypeptides that are contiguous amino acids, or between 10 and 650, including any number not specifically described herein, as described herein Numbers up to the total number of amino acids in the full-length protein are included (for example, up to 144 in SEQ ID NO: 2, up to 123 in SEQ ID NO: 4). Such biologically active moieties can be prepared by recombinant methods and can be evaluated for one or more functional activities in native carbohydrate utilization-related proteins or multidrug transporter proteins. . As used herein, a fragment includes at least 5 consecutive amino acids in any of the even numbered sequences of SEQ ID NOs: 2-364. However, the present invention encompasses other fragments, any fragment larger than 6, 7, 8 or 9 amino acids in any of the above proteins.

  The present invention encompasses various nucleotide amino acid sequences. “Variant” means a sequence that is sufficiently identical. Therefore, the present invention provides an isolated nucleic acid sufficiently identical to the nucleotides encoding the carbohydrate-related utilization protein and the multidrug transporter protein represented by even numbers in SEQ ID NOs: 2 to 364, or SEQ ID NOs: 1 to 363. Among them, nucleic acids that hybridize under stringent conditions with nucleic acids indicated by odd numbers or their complementary sequences are included. Variants further include polypeptides encoded by the nucleotide sequence variants of the present invention. Furthermore, the polypeptide of the present invention has an amino acid sequence sufficiently identical to the amino acid sequence represented by the even number among SEQ ID NOs: 2 to 364. “Sufficiently identical” means that the first amino acid sequence or nucleotide sequence has a sufficient or minimal number of corresponding or identical amino acid residues compared to the second amino acid sequence or nucleotide sequence, whereby Meaning that a common structural domain is shown and / or a common functional activity is envisaged. Conserved variants include sequences that differ due to the degeneracy of the genetic code.

  In general, at least 45%, 55% or 65% identity with either the even numbered amino acid sequence of SEQ ID NOs: 2-364 or the nucleotide sequence of odd numbered SEQ ID NO: 1-363, respectively. , Preferably at least about 70% or 75% identity, more preferably at least about 80%, 85% or 90%, most preferably at least about 91%, 92%, 93%, 94%, 95%, 96 Amino acid or nucleotide sequences having%, 97%, 98% or 99% sequence identity are defined herein as being sufficiently identical. The protein variants encompassed by the present invention are biologically active and retain the desired biological activity in the native protein, such a desired activity being a carbohydrate utilization-related activity or multidrug transporter as described herein Active. In biologically active variants of the protein of the invention, the protein is 1-15, 1-10, such as 6-10, 5, 4, 3, 2, or simply 1 The amino acid residues are different.

  Within the population (eg Lactobacillus acidophilus population) there are also naturally occurring variants. Such variants can be identified by well-known molecular biological techniques such as PCR and hybridization described below. Variants also include nucleotide sequences obtained by synthesis, for example, sequences generated by site-directed mutagenesis or PCR-mediated mutagenesis and still encode carbohydrate utilization-related proteins or multidrug transporter proteins . One or more nucleotide or amino acid substitutions, additions or deletions are introduced into the nucleotide or amino acid sequences disclosed herein, thereby substituting, adding or deleting the encoded protein. Can be introduced. The addition (insertion) or deletion (truncation) may be performed at the N-terminal or C-terminal of the natural protein, or may be performed at one or more sites in the natural protein. Similarly, one or more nucleotide or amino acid substitutions may be made at one or more sites in the native protein.

  For example, conservative amino acid substitutions may be made at one or more predicted amino acid residues, preferably non-essential amino acid residues. A “non-essential” amino acid residue is a residue that, when changed in the wild-type sequence of a protein, does not change the biological activity of the protein, whereas an “essential” amino acid contributes to biological activity. A necessary amino acid. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains are known in the art. These families include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, Tyrosine, cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg threonine, valine, isoleucine), and aromatic side chains ( For example, amino acids having tyrosine, phenylalanine, tryptophan, histidine) are included. If the amino acid residue is a conserved amino acid residue or an amino acid residue within a conserved motif and is essential for protein activity, no substitution is performed.

  Alternatively, all or part of the entire length of the carbohydrate utilization-related coding sequence or the multidrug transporter coding sequence can be randomly mutated by saturation mutagenesis or the like. Mutants can be expressed by recombination and assayed using standard assays to screen for mutants that retain carbohydrate utilization-related activity or multidrug transporter activity. Mutagenesis and methods for altering nucleotide sequences are known in the art. For example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492, Kunkel et al. (1987) Methods in Enzymol. Molecular Biology (MacMillan Publishing Company, New York), and references in these references. checking ... Of course, mutations in the DNA encoding the variant must not impair the reading frame, nor should it result in a complementary region that can generate a secondary mRNA structure. See European Patent Application Publication No. 75444. For guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest, see Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

  Deletions, insertions, and substitutions in protein sequences encompassed by the present invention do not fundamentally change the properties of such proteins. However, those skilled in the art will appreciate that if it is difficult to accurately predict in advance the effects of substitutions, deletions or insertions, the effects can be assessed by routine screening assays. That is, the activity can be evaluated by comparing the activity of the changed sequence with the activity of the original sequence. See the “Usage” section below for examples of assays used to measure carbohydrate utilization related activity or multidrug transporter activity.

The nucleotide sequence variants and amino acid sequence variants of the present invention also include sequences obtained by mutagenesis methods such as DNA shuffle and recombinant production methods. In such a method, the coding region of one or more different carbohydrate utilization-related proteins or multidrug transporter proteins is used to create a novel carbohydrate utilization-related protein or novel multidrug transporter protein having the desired properties. Can be used. By such methods, a library of recombinant polynucleotides is generated from a related sequence polynucleotide population comprising sequence regions that have substantial sequence identity and are capable of homologous recombination in vitro or in vivo. For example, by using this approach, a target domain is encoded between the carbohydrate utilization-related gene or multidrug transporter gene of the present invention and other known carbohydrate utilization-related genes or multidrug transporter genes. shuffling sequence motifs, such as increased K _m in the case of enzymes, the new gene can be obtained characteristics of interest encodes a protein that enhanced. Such DNA shuffle strategies are known in the art. For example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751, Stemmer (1994) Nature 370: 389-391, Crameri et al. (1997) Nature Biotech. 15: 436-438, Moore et al. (1997) J. Mol. Biol. 272: 336-347, Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509, Crameri et al. (1998) Nature 391: 288 -291, and U.S. Pat. Nos. 5,605,793 and 5837458.

  Variants of carbohydrate utilization related proteins and multidrug transporter proteins can function as agonists (mimetics) or antagonists. A protein agonist can retain substantially the same activity or a subset of activities as the native biological activity of the protein. An antagonist of a protein is one of the natural activities of such proteins by, for example, competitively binding to a member present downstream or upstream of a cellular signaling cascade including a carbohydrate utilization-related protein or a multidrug transporter protein. Or more than one can be inhibited.

  Variants of carbohydrate utilization-related proteins or multidrug transporter proteins that function as either agonists or antagonists are combinatorial libraries of carbohydrate utilization-related proteins or variants of multidrug transporter proteins (eg, truncation variants) It can be identified by screening for agonist or antagonist activity. In one embodiment, a diversified library of carbohydrate utilization related variants is generated by combinatorial mutagenesis at the nucleic acid level and encoded by a diversified gene library. A diversified library of carbohydrate utilization-related or multidrug transporter variants is a degenerate set of sequences that may be carbohydrate utilization-related or multidrug transporter sequences, as individual polypeptides. Alternatively, a mixture of synthetic oligonucleotides, eg, gene sequences, so that it can be expressed as a larger set of fusion proteins (eg, for phage display) containing carbohydrate utilization-related sequences or sets of multidrug transporter sequences therein Can be produced by enzymatic ligation. There are many ways to generate a library of potential carbohydrate utilization-related or multidrug transporter variants from degenerate oligonucleotide sequences. Chemical synthesis of the degenerate gene sequence is performed with an automatic DNA synthesizer, and then the synthesized gene is linked to an appropriate expression vector. By using a degenerate set of genes, it is possible to provide the entire sequence encoding the desired set of potential carbohydrate utilization related sequences or multidrug transporter sequences as a single mixture. Methods for synthesizing degenerate oligonucleotides are known in the art (eg, Narang (1983) Tetrahedron 39: 3; Itakura et al. (1984) Annu. Rev. Biochem. 53: 323; Itakura et al. ( 1984) Science 198: 1056; Ike et al. (1983) Nucleic Acid Res. 11: 477).

  Furthermore, in order to produce various groups of carbohydrate utilization-related fragments or multidrug transporter fragments used for screening and selection of variants of carbohydrate utilization-related proteins or multidrug transporter proteins, carbohydrate utilization-related proteins or multidrugs A library of fragments of the transporter protein coding sequence can also be used. In one embodiment, a library of coding sequence fragments can be generated by the following method. That is, under conditions such that nicking occurs only once per molecule, a double-stranded PCR fragment of a carbohydrate utilization-related coding sequence or a multidrug transporter coding sequence is treated with a nuclease to denature the double-stranded DNA, The DNA is renatured to form a double stranded DNA that can contain sense / antisense pairs in different nicking products, and S1 nuclease treatment removes the single stranded portion from the reshaped double stranded molecule. The fragment library obtained as a result can be prepared by linking to an expression vector. By this method, an expression library encoding N-terminal fragments and internal fragments of various sizes of carbohydrate utilization-related proteins or multidrug transporter proteins can be obtained.

  Several methods are known in the art for screening gene products from combinatorial libraries created by point mutations or truncations, and for screening cDNA libraries to obtain gene products with selected characteristics. It has been. Such a technique can be used for high-speed screening of gene libraries created by inducing combinatorial mutations in carbohydrate utilization-related proteins or multidrug transporter proteins. The most widely used approach to screening large gene libraries involves cloning the gene library into a replicable expression vector, transforming appropriate cells with the resulting library of vectors, Then, by detecting the desired activity, the combinatorial gene is expressed under conditions that facilitate the isolation of the vector encoding the gene of the detected gene product. It is. Recursive ensemble mutagenesis (REM) is a technique that increases the frequency of functional variants in a library, but this technique is used to identify carbohydrate utilization-related variants or multidrug transporter variants. Can be used in combination with screening assays (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave et al. (1993) Protein Engineering 6 (3): 327-331) .

Sequence identity Glucose utilization related sequences and multidrug transporter sequences belong to a family of molecules with conserved functional features. “Family” means two or more proteins or nucleic acids whose nucleotide and amino acid sequences are sufficiently identical. Families with a number of very different groups are classified into subfamilies. A clan is a group of families that are considered to have a common ancestor. The members of the tribe often have similar tertiary structures. “Sequence identity” refers to nucleotide or amino acid residues that are identical when the two sequences are aligned so that there is a maximum of match in at least one particular comparison window. “Comparison window” means a contiguous segment for optimal alignment of two nucleotide or amino acid sequences, wherein the second sequence is added or deleted (ie, a gap) from the first sequence. ) May be present. Typically, the comparison window in a nucleic acid alignment is a continuous length of at least 20 nucleotides, and can optionally be 30, 40, 50, 100, or even longer. The comparison window in an amino acid sequence alignment is a length that is at least 6 consecutive amino acids in length and can optionally be 10, 15, 20, 30, or even longer. Those of ordinary skill in the art recognize that a gap penalty is usually introduced and the gap penalty is subtracted from the number of matches to avoid increasing the similarity by including a gap.

  Family members may be of the same or different species and include not only different proteins but also homologous proteins. Family members often exhibit common functional characteristics. Isolation of homologues is disclosed below using cDNA or portions thereof as hybridization probes based on the identity to carbohydrate-utilizing nucleic acid sequences derived from Lactobacillus acidophilus or multidrug transporter nucleic acid sequences disclosed herein. Can be performed according to standard hybridization methods under stringent hybridization conditions.

  An alignment is performed to determine the percent identity of two amino acid sequences or nucleotide sequences. The identity ratio of the two sequences is a function of the number of identical residues shared by the two sequences in the comparison window (ie, identity ratio = number of identical residues / total number of residues X100). In one embodiment, these sequences are the same length. A method similar to that described below can be used to determine the percent identity between two sequences. These methods can be used with or without considering the gap. By inspecting, it is also possible to prepare an alignment in a manual manner.

  If the amino acid sequence difference is due to a conservative substitution, the ratio of identity can be adjusted upward to make corrections that take into account the conservative nature of the substitution. Methods for making this adjustment are known in the art. Usually, conservative substitutions are calculated as partial mismatches rather than complete mismatches, thus increasing the percent sequence identity.

  A mathematical algorithm can be used to determine the percent identity of two sequences. Non-limiting examples of mathematical algorithms include Karlin and Altschul (1990) Proc. Natl. Acad., Modified in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877. Sci. USA 87: 2264 algorithm, Myers and Miller (1988) CABIOS 4: 11-17 algorithm, Smith et al. (1981) Adv. Appl. Math. 2: 482 local alignment algorithm, Needleman and Wunsch (1970 ) J. Mol. Biol. 48: 443-453 global alignment algorithm and Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444-2448 similarity search (search-for-similarity- method).

  Various computer implementations based on these mathematical algorithms are designed to allow determination of sequence identity. The BLAST program of Altschul et al. (1990) J. Mol. Biol. 215: 403 is based on the algorithm of Karlin and Altschul (1990) described above. A search to obtain a nucleotide sequence homologous to the nucleotide sequence of the present invention can be performed using the BLASTN program with a score = 100 and a word length = 12. To obtain an amino acid sequence homologous to the sequence encoding the protein or polypeptide of the present invention, the BLASTX program can be used with a score = 50 and a word length = 3. Obtain an alignment that takes into account the gap by using gapped BLAST (in BLAST 2.0), as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Can do. PSI-BLAST can be used to detect distant relationships between molecules. See Altschul et al. (1997) above. The default parameters of each program can be used for all BLAST programs. By inspecting, it is also possible to prepare an alignment in a manual manner.

  Another program that can be used to determine percent sequence identity is the ALIGN program (version 2.0), which uses the mathematical algorithm of Myers and Miller (1988) described above. . When comparing amino acid sequences, the ALIGN program can use the PAM120 weight residue table, gap length penalty 12, and gap penalty 4.

  In addition to the ALIGN and BLAST programs, the BESTFIT, GAP, FASTA, and TFASTA programs are available from GCG Wisconsin Genetics Software Package, Version 10 (Accelrys, Inc., as part of 9685 Scranton Rd., San Diego, California, USA). Yes, and can be used to perform sequence alignment. A preferred program is GAP version 10, which uses the above-mentioned Needleman and Wunsch (1970) algorithm. Unless otherwise specified, the sequence identity levels presented herein are the values obtained using GAP version 10 with the following parameters: That is, the nucleotide sequence identity% and similarity% include nwsgapdna., With a gap weight of 50 and a gap length weight of 3. The cmp scoring matrix is used, and the BLOSUM62 scoring matrix is used with a gap weight of 8 and a gap length weight of 2 for% identity and similarity of amino acid sequences. An “equivalent program” is the same nucleotide or amino acid residue match and the same percentage (%) of identity between any two sequences of interest compared to the equivalent alignment created with GAP version 10. It refers to any sequence comparison program that creates an alignment.

  A sequence alignment on a database created by an algorithm such as BLASTIN, FASTA, or BLASTP for a search sequence is generally described as a “hit”. A hit against one or more database sequences created from the search sequence by BLASTNFASTA, BLASTP or similar algorithm identifies and aligns similar portions in the sequence. A hit against a database sequence generally represents an overlap of fragments of the sequence length of the search sequence (part of the sequence subjected to the search or a fragment). However, such duplication can represent the full length of the search sequence. Hits in the alignment of search sequences created by BLASTN, FASTA or BLASTP algorithms against sequences on the database are typically placed in order of similarity and length of overlapping portions of the sequence. .

  Polynucleotide and polypeptide hits aligned by the BLASTN, FASTA, or BLASTP algorithm against the search sequence yield an “Expect” value. The expected value (E value) indicates the number of hits that can be “expected” to search a predetermined number of consecutive arrays at random when searching a database of a predetermined size. The expected value is used as a significant threshold to see if hits against a database such as the GenBank or EMBL database reflect actual similarities. For example, an E value of 0.1 given for polynucleotide hits means that in a database of the same size as the GenBank database size, there will be a random match of 0.1 in aligned parts with similar scores. Means you can expect. According to this criterion, there is a 90% chance that the aligned and matched portions in the polynucleotide sequence are identical. For sequences that have an E value of 0.01 or less in the aligned and matched portions, the chance of a random match being found in the GenBank database using the BLASTN or FASTA algorithm is less than 1%.

  According to an embodiment of the present invention, the “variant” of the polynucleotide and polypeptide of the present invention includes a sequence having an E value of about 0.01 or less compared to the polynucleotide sequence or polypeptide sequence of the present invention. It is. That is, the E value measured using the BLASTN, FASTA, or BLASTP algorithm with the parameters described herein is 0.01 or less and may be the same as the polynucleotide or polypeptide of the present invention. All sequences that are 99% or more become polynucleotide and polypeptide variants. In another embodiment, the E value measured using the BLASTN or FASTA algorithm with the parameters described herein is less than 0.01 and is 99% likely to be identical to the polynucleotide of the invention. The above-described sequences of the present invention and sequences having the same or less number of nucleic acids are polynucleotide variants. Similarly, the E value measured using the BLASTP algorithm with the parameters described herein is 0.01 or less, and the possibility of being the same as the polypeptide of the present invention is 99% or more. A sequence having the same or fewer amino acids than a polypeptide is a variant of the polypeptide.

  As described above, percent identity is determined by aligning sequences using the BLASTN, FASTA, or BLASTP algorithm with the running parameters described herein, identifying the number of identical nucleic acids or amino acids in the aligned portion, The number of nucleic acids or amino acids is determined by dividing by the total number of nucleic acids or amino acids in the polynucleotide or polypeptide sequence of the invention and multiplying by 100 to determine the percent identity. For example, for a polynucleotide of the invention having 220 nucleic acids, in a GenBank database with 520 nucleic acids, for a range of 23 nucleotides in an alignment created by the BLASTN algorithm using the parameters described herein The polynucleotide sequence is hit. The 23 nucleotides hit are 21 identical nucleotides, one gap, and one different nucleotide. Therefore, the identity ratio of the polynucleotide of the present invention to the GenBank library is 21/220 × 100, that is, 9.5%. Thus, the polynucleotide sequence on the GenBank database is not a variant of the polynucleotide of the present invention.

(Identification and isolation of homologous sequences)
Carbohydrate utilization-related nucleotide sequences identified on the basis of sequence identity to the carbohydrate utilization-related nucleotide sequences or multidrug transporter nucleotide sequences described herein, or fragments and variants thereof, are also encompassed by the present invention. . For example, methods such as PCR or hybridization can be used to identify sequences substantially identical to the sequences of the invention from a cDNA or genomic library. For example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York) and Innis, et al., (1990) PCR Protocols: A Guide to Methods and Applications ( See Academic Press, .New York). Methods for constructing such cDNA and genomic libraries are generally known in the art and are also disclosed in the above references.

In the hybridization method, the hybridization probe may be a genomic DNA fragment, cDNA fragment, RNA fragment, or other oligonucleotide, and may be all or part of the known nucleotide sequences disclosed herein. . Furthermore, they may be labeled with a detectable group such as ³² P, or any other detectable marker such as other radioisotopes, fluorescent compounds, enzymes, or enzyme cofactors. Probes for hybridization can be produced by labeling synthetic oligonucleotides based on the carbohydrate-related nucleotide sequences or multidrug transporter nucleotide sequences disclosed herein. In addition, use a degenerate primer designed based on a nucleotide residue or amino acid residue conserved in a known carbohydrate utilization-related nucleotide sequence or multidrug transporter nucleotide sequence, or an amino acid sequence encoded by the nucleotide sequence. Can do. Hybridization probes include at least about 10, preferably about 20, more preferably 50, 75, 100, 125, 150, 175, 200, 250, consecutive in the nucleotide sequence of the invention, or fragments or variants thereof. Nucleotide sequence regions that hybridize under stringent conditions to 300, 350, or 400 nucleotides are usually included. In order to achieve specific hybridization under various conditions, such probes include sequences having characteristics common to carbohydrate utilization-related protein sequences or multidrug transporter protein sequences. It is. Probe preparations for hybridization are generally known in the art and are incorporated herein by reference, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

  In one embodiment, the complete nucleotide sequence encoding a carbohydrate utilization protein or multidrug transporter protein is used to identify novel carbohydrate utilization related or multidrug transporter sequences and mRNA. In another embodiment, a probe is a fragment of a nucleotide sequence disclosed herein. In some embodiments, the length of the nucleotide sequence that hybridizes to the probe under stringent conditions is at least 300, 325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800. 900, 1000, 1200, 1400, 1600, 1800 or 2000 nucleotides.

  Substantially identical sequences hybridize to each other under stringent conditions. The “stringent condition” means a condition in which the detection level is higher when the probe hybridizes to the target sequence than when it hybridizes to other sequences (for example, at least twice the background). Usually, stringent conditions include hybridization and wash conditions such that nucleotides having at least about 60%, 65%, 70%, preferably 75% sequence identity are usually in a hybridized state with each other. Is included. Stringent conditions are known in the art and can be found in Current Protocols in Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6. Hybridization generally occurs within about 24 hours, usually between about 4 and about 12 hours.

  Stringent conditions are sequence-dependent and will be different in different circumstances. In order to obtain homologs and orthologs encompassed by the present invention, the full length of a nucleic acid sequence or a partial nucleic acid sequence may be used. “Ortholog” means a gene that is derived from a common ancestor gene and found in different species as a result of species speciation. Genes found in different species are orthologous if their nucleotide sequences and / or the protein sequences encoded by them are the same as the substantial identity as defined herein. Conceivable. Ortholog functions are often highly conserved among species.

  The stringent conditions when using a probe include a salt concentration of less than about 1.5M Na ion, usually pH 7.0-8.3, about 0.01-1.0M Na ion concentration (or other The temperature conditions are at least about 30 ° C. for short probes (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes (eg, greater than 50 nucleotides).

Washing after hybridization is a means of controlling specificity. Two important factors are the ionic strength and temperature of the final wash solution. For detection of a sequence that hybridizes to a full-length or nearly full-length target sequence, the temperature under stringent conditions is about the melting temperature (T _m ) of the specific sequence at a given ionic strength and pH. Select 5 ° C lower. However, stringent conditions, depending on the desired stringency separately determined herein, include 20 ° C. lower temperature range of 1 ℃ than T _m. For DNA-DNA hybrids, the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138: 267-284: T _m = 81.5 ° C. + 16.6 (log M) +0.41 (% GC) − (0.61 ( % Form) -500 / L can be used to determine _Tm ; where M is the molar concentration of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% Form is the proportion of formamide in the hybridization solution, and L is the length of the hybrid in base pairs T _m is the probe in which 50% of the complementary target sequence is perfectly matched Is the temperature (under a specific ionic strength and pH) to hybridize to

By changing the stringency of hybridization conditions and / or washing conditions, it is possible to obtain detection ability for sequences having different degrees of homology. Targeting 100% identical sequences (homologous probing) requires stringency conditions that do not cause mismatches. By allowing mismatches in nucleotide residues to occur, sequences with a low degree of similarity can be detected (heterologous probing). For each 1% mismatch, the _Tm is reduced by about 1 ° C., and thus hybridization and / or wash conditions can be manipulated so that sequences with the targeted identity ratio hybridize. For example, if the preferred sequence having 90% or more sequence identity, the T _m it is sufficient to decrease 10 ° C.. Two nucleotide sequences can be substantially identical without hybridizing to each other under stringent conditions if the polypeptides they encode are substantially identical. Such a situation can occur, for example, when replicating a nucleic acid using the maximum codon degeneracy of the genetic code.

  Examples of low stringency conditions include hybridization using 30-35% formamide, 1M NaCl, 1% SDS (sodium dodecyl sulfate), 37 ° C. buffer solution, 1 × SSC to 2 × SSC (20 × Washing at 50-55 ° C. with SSC = 3.0 M NaCl / 0.3 M trisodium citrate) is included. Examples of moderate stringency conditions include 40% to 45% formamide, 1.0 M NaCl, 1% SDS, hybridization at 37 ° C, and 55 ° C to 60 ° C in 0.5x to 1xSSC. Cleaning is included. Examples of high stringency conditions include 50% formamide, 1M NaCl, 1% SDS, hybridization at 37 ° C., and washing at 60-65 ° C. with 0.1 × SSC. If desired, the wash buffer may comprise about 0.1% SDS to about 1% SDS. Usually, the hybridization time is generally less than 24 hours, usually about 4 to about 12 hours. Guidelines for nucleic acid hybridization can be found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); Ausubel et al., Eds. (1995) Current Protocols. in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et at (1989) Molecular Cloning: A Laboratory Manual (2d ed .: Cold Spring Harbor Laboratory Press, Plainview, New York).

  In the PCR approach, oligonucleotide primers are designed and used in a PCR reaction to amplify the corresponding DNA sequence from cDNA or genomic DNA extracted from the target organism. PCR primers are preferably at least about 10 nucleotides in length and most preferably at least about 20 nucleotides in length. Methods for designing PCR primers and methods for PCR cloning are generally known in the art, and Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York). Innis et al., Eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press: New York); and Innis and Gelfand eds. (1999) See also PCR Methods Manual (Academic Press, New York). Known PCR methods include primer pairs, nested primers, single specific primers, degenerate primers, gene specific primers, vector specific primers, primers with partial mismatches, etc. However, it is not limited to these.

(Assay)
Disclosed are diagnostic assays that detect the expression of polypeptides and / or nucleic acid molecules disclosed in the present invention in a sample and the disclosed activity in the sample. Examples of methods for detecting the presence or absence of a disclosed nucleic acid or a protein comprising a disclosed polypeptide in a sample include food products / dairy products / feed products, starter cultures (mother, seed, bulk / set, concentrated, dried, Freeze-drying, freezing), cultivated food product / dairy product / feed, supplement, bioprocessed fermented material, or subject that has received a probiotic material, and the sample contains the disclosed poly A peptide or nucleic acid (eg, mRNA or genomic DNA containing the disclosed nucleic acid, or fragment thereof) is contacted with a compound or agent capable of detecting, and the presence or absence of the disclosed sequence is detected. Results from a sample obtained from a food, supplement, culture, product, or subject can be compared to results from a sample obtained from the culture, product, or subject as a control.

  One agent for detecting mRNA or genomic DNA comprising a nucleotide sequence disclosed in the present invention is a labeled nucleic acid probe that can hybridize to the disclosed nucleotide sequence of mRNA or genomic DNA. For example, the nucleic acid probe is a disclosed nucleic acid represented by an odd number in SEQ ID NOs: 1 to 363, or a disclosed nucleic acid such as a part thereof. The portion is, for example, at least 15, 30, 50, 100, 250 or 500 nucleotides long and is sufficient to specifically hybridize under stringent conditions with mRNA or genomic DNA comprising the disclosed nucleic acid sequences. It is an important part. Suitable probes for use in the diagnostic assays of the invention are described herein.

One agent for detecting a protein comprising the disclosed polypeptide sequence is an antibody capable of binding to the disclosed polypeptide, preferably a detectably labeled antibody. The antibody may be polyclonal, but more preferably monoclonal. An intact antibody, or a fragment thereof (eg, Fab or F (ab ′) ₂ ) can be used. With respect to a probe or antibody, the term “labeled” implies directly labeling the probe or antibody by attaching (ie, physically linking) a detectable substance to the probe or antibody; It also implies indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.

  The term “sample” includes tissues, cells, and biological fluids that are present in or isolated from a subject, and further have, or have, cells of a starter culture, or such culture. Foods obtained using such cultures are also included. That is, the detection method of the present invention can be used to detect mRNA, protein, or genomic DNA containing a disclosed sequence in a sample both in vitro and in vivo. In vitro methods for detecting mRNA comprising the disclosed sequences include Northern hybridization and in situ hybridization. In vitro methods for detecting proteins comprising the disclosed polypeptides include enzyme linked immunosorbent assays (ELISA), Western blots, immunoprecipitation reactions, and immunofluorescence. In vitro methods for detecting genomic DNA containing the disclosed nucleotide sequences include Southern hybridization. Further, in vivo methods for detecting proteins comprising the disclosed polypeptides include introducing labeled antibodies against the disclosed polypeptides into the subject. For example, the antibody can be labeled with a radioactive marker, and its presence and location in a subject can be detected by standard imaging techniques.

  In one embodiment, the sample contains protein molecules from a test subject that has consumed a probiotic material. Alternatively, the sample may contain mRNA or genomic DNA of the starter culture.

  The present invention also includes a kit for detecting the presence or absence of a disclosed nucleic acid or a protein containing the disclosed polypeptide in a sample. Such kits can be used to determine whether a microorganism that expresses a particular polypeptide of the invention is present in a food or starter culture, or in a subject that has consumed a probiotic substance. . For example, the kit may comprise a labeled compound or agent capable of detecting the disclosed polypeptide or mRNA in the sample and a means for quantifying the disclosed polypeptide in the sample (eg, the disclosed polypeptide). Or an oligonucleotide probe that binds to, for example, DNA encoding an even-numbered sequence of SEQ ID NOs: 2-364). The kit may be accompanied by detailed instructions on the use of such compounds.

  In the case of antibody-based kits, such kits include, for example, (1) a primary antibody that binds to the disclosed polypeptide (eg, bound to a solid support) and, optionally, (2) the disclosed polypeptide. Or it may contain a secondary antibody different from the primary antibody that binds to the primary antibody and binds to the detectable agent. In the case of oligonucleotide-based kits, such kits may, for example, (1) oligonucleotides that hybridize to the disclosed nucleic acid sequences, such as detectably labeled oligonucleotides, or (2) amplify the disclosed nucleic acids. A pair of primers useful for may be included.

  The kit may include, for example, a buffer, a preservative, or a protein stabilizer. The kit may include the components necessary to detect a detectable agent (eg, an enzyme or substrate). The kit may also include a control sample or series of control samples that are subjected to the assay and compared to the test sample. Each component of the kit is typically contained in an individual container, all of which are in a single package with instructions for use.

  In one embodiment, the kit includes a plurality of probes in an array format, such as, for example, those described in US Pat. Nos. 5,420,087 and 5,455,531, and WO 95/00530, incorporated herein by reference. Probes for use in the array can be synthesized directly on the surface of the array, as disclosed in WO95 / 00530, or can be synthesized prior to immobilization on the surface of the array (Gait, ed. (1984) Oligonucleotide Synthesis a Practical Approach IRL Press, Oxford, England). The probe can be immobilized on the surface using techniques well known to those skilled in the art, such as those described in US Pat. No. 5,420,087. The probe may be a nucleic acid sequence or a peptide sequence (preferably purified), or an antibody.

  Arrays can be used to screen organisms, samples, or products for differences in the content of their genomic cDNA, polypeptide, or antibody, such as the presence or absence of specific sequences or proteins, and the concentration of those substances. it can. For example, binding to a capture probe is detected by a signal generated from a label bound to a nucleic acid molecule containing the disclosed nucleic acid sequence, a label bound to a polypeptide containing the disclosed amino acid sequence, or a label bound to an antibody. The method includes contacting a molecule comprising a disclosed nucleic acid, polypeptide, or antibody with a first array having a plurality of capture probes and a second array having another plurality of capture probes. Each hybridization result can be compared to analyze the difference in expression between the first sample and the second sample. The first plurality of capture probes can be from a control sample, eg, from a wild-type lactic acid bacterium, or from a control subject, eg, a food, nutritional supplement, starter culture sample, or body fluid. The second plurality of capture probes may be obtained from an experimental sample, such as one obtained from a mutant lactic acid bacterium, or from a subject that has consumed a probiotic substance, such as a starter culture sample or biological fluid.

  These assays are particularly useful for microbial selection and quality control operations that require the detection of unwanted materials. Detection of specific nucleotide sequences or polypeptides is useful for determining the genetic composition of food, fermentation products or industrial microorganisms, or for determining microorganisms present in the digestive system of animals or humans that have ingested probiotics.

(Antisense nucleotide sequence)
The invention also encompasses antisense nucleic acid molecules, ie, molecules complementary to a sense nucleic acid encoding a protein, eg, molecules complementary to the coding strand of a double stranded cDNA molecule, or molecules complementary to an mRNA sequence. . Accordingly, an antisense nucleic acid can hydrogen bond with a sense nucleic acid. Antisense nucleic acids are complementary to all or part of the coding strand of a carbohydrate utilization-related or multidrug transporter (eg, all or part of a protein coding region (or open reading frame)). An antisense nucleic acid may be antisense to a non-coding region of the coding strand of a nucleotide sequence encoding a carbohydrate utilization related protein or a multidrug transporter protein. Non-coding regions are 5 ′ and 3 ′ sequences that flank the coding region and are not translated into amino acids. Antisense nucleotide sequences are useful to impair target gene expression. Antisense constructs with 70%, preferably 80%, more preferably 85%, 90%, 95% sequence identity to the corresponding sequence can be used.

  Based on the coding chain sequence (for example, the even numbered sequence in SEQ ID NOs: 2 to 364) encoding a carbohydrate utilization-related protein or multidrug transporter protein disclosed herein, the Watson and Crick base pairing rule Thus, the antisense nucleic acid of the present invention can be designed. The antisense nucleic acid may be complementary to the entire coding region of carbohydrate utilization-related or multidrug transporter mRNA, but the coding region or non-coding region of carbohydrate utilization-related mRNA or multidrug transporter mRNA Oligonucleotides that are antisense to only a portion of are more preferred. For example, the antisense oligonucleotide may be complementary to a region in the vicinity of the translation initiation site in carbohydrate utilization-related mRNA or multidrug transporter mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or 100 nucleotides, 200 nucleotides in length, or It can be even longer. The antisense nucleic acid of the present invention can be constructed using chemical synthesis methods and enzyme ligation methods known in the art.

  For example, antisense nucleic acids (eg, antisense oligonucleotides) use natural nucleotides, increase the biostability of a molecule, or increase the physical stability of a double stranded formed between an antisense nucleic acid and a sense nucleic acid. It can be obtained by chemical synthesis by using variously modified nucleotides to increase. Such modified nucleotides include, but are not limited to, phosphorothioate derivatives and acridine substituted nucleotides. Alternatively, an antisense nucleic acid (eg, an antisense oligonucleotide) is a naturally occurring nucleotide or, but not limited to, for example, to increase the biological stability of a molecule or It can be chemically synthesized using variously modified nucleotides to increase the physical stability of the double-stranded molecule formed with the sense nucleic acid. Alternatively, antisense nucleic acids can be made biologically using an expression vector in which the nucleic acid is subcloned in the antisense direction (ie, RNA transcribed from the inserted nucleic acid is the target) The target nucleic acid is in the direction of antisense).

  The antisense nucleic acid of the present invention may be an α-anomeric nucleic acid molecule. α-anomeric nucleic acids form specific double-stranded hybrids with complementary RNAs, and these double strands are parallel to each other unlike normal β-units (Gaultier et al. (1987) Nucleic Acids Res. 15: 6625-6641). Antisense nucleic acid molecules are 2′-o-methyl ribonucleotides (Inoue et al. (1987) Nucleic Acids Res. 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al. (1987) FEBS Lett. 215: 327-330).

  The present invention also includes ribozymes. A ribozyme is a catalytic RNA molecule having a ribonuclease activity that cleaves single-stranded nucleic acid such as mRNA having a ribozyme complementary region. Ribozymes, such as hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334: 585-591), cleave carbohydrate-related mRNA transcripts by catalysis, thereby providing carbohydrate utilization-related mRNAs or mRNAs. Inhibits translation of A ribozyme having specificity for a nucleic acid encoding a carbohydrate utilization-related protein or a nucleic acid encoding a multidrug transporter protein is a nucleotide sequence of a carbohydrate utilization-related cDNA or a multidrug transporter cDNA disclosed in the present specification. It can be designed based on (for example, an odd numbered array of SEQ ID NOs: 1 to 363). See, for example, US Pat. No. 4,987,071, and US Pat. No. 5,116,742. Alternatively, carbohydrate utilization-related mRNA or multidrug transporter mRNA can be used to select a catalytic RNA having specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel and Szostak (1993) Science 261: 1411-1418.

  The invention also encompasses nucleic acid molecules that form triple helix structures. For example, a carbohydrate utilization-related gene expression or a multidrug transporter gene expression is a regulatory region of a carbohydrate utilization-related protein or a multidrug transporter protein (eg, a carbohydrate utilization-related or multidrug transporter promoter and / or enhancer). Can be inhibited by forming a triple helix structure that prevents transcription of a carbohydrate utilization-related gene or a multidrug transporter gene in the target cell by targeting a nucleotide sequence complementary to. See Helene (1991) Anticancer Drug Des. 6 (6): 569: Helene (1992) Ann. N Y. Acad Sci. 660: 27; and Maher (1992) Bioassays 14 (12): 807.

  In some embodiments, the nucleic acid molecules of the invention may be modified at the base moiety, sugar moiety, or phosphate backbone, eg, to enhance the stability, hybridization, or solubility of the molecule. For example, the nucleic acid deoxyribose phosphate backbone can be modified to produce peptide nucleic acids (see Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4: 5). As used herein, the term “peptide nucleic acid” or “PNA” is a nucleic acid mimic in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only four natural nucleobases are retained. Refers to, for example, a DNA mimic. The neutral backbone of PNA has been shown to allow specific hybridization to DNA and RNA under low ionic strength conditions. The synthesis of PNA oligomers is described, for example, by the standard solid phase peptide described in Hyrup et al. (1996); Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci: USA 93: 14670. Can be performed using a synthesis protocol.

  PNA can be used as an antisense agent or an antigene agent to effect sequence-specific regulation of gene expression, for example, by inducing transcriptional or translational arrest or by suppressing replication. The PNA of the present invention can be used, for example, by a PCR clamp method targeting PNA; as an artificial restriction enzyme when used in combination with other enzymes such as S1 nuclease (Hyrup (1996), ibid); or for DNA sequences and hybridization As a probe or primer (Hyrup (1996), Perry-O'Keefe et al. (1996)), for example, for analysis of single base pair mutations in a gene.

  In another embodiment, the carbohydrate utilization-related molecule or multidrug transporter molecule PNA can be modified to promote its stability, specificity, or cellular uptake. Such modifications can be made by attaching lipophilic groups or other helper groups to PNA, or by forming PNA-DNA chimeras, using liposomes, or other drugs known in the art. This can be done by delivery means. The synthesis of PNA-DNA chimeras is described in Hyrup (1996); Finn et al. (1996) Nucleic Acids Res. 24 (17): 3357-63; Mag et al. (1989) Nucleic Acids Res. 17: 5973; Peterson et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119.

(Fusion protein)
The present invention also includes carbohydrate-related or multidrug transporter chimeric or fusion proteins. A “chimeric protein” or “fusion protein” of a carbohydrate utilization-related or multidrug transporter is a polysaccharide of a carbohydrate utilization-related or multidrug transporter operably linked to a polypeptide that is not a carbohydrate utilization-related or multidrug transporter. Contains peptides. A “carbohydrate utilization-related or multidrug transporter polypeptide” is a polypeptide having an amino acid sequence corresponding to a carbohydrate utilization-related or multidrug transporter protein. A polypeptide that is not a multidrug transporter is a polypeptide having an amino acid sequence corresponding to a protein that is not substantially identical to a carbohydrate utilization-related or multidrug transporter protein, and is derived from the same organism or a different organism Refers to a polypeptide. Within the carbohydrate utilization-related or multidrug transporter fusion protein, the carbohydrate utilization-related or multidrug transporter polypeptide may correspond to the entire carbohydrate utilization-related or multidrug transporter protein. It may correspond to that part, but preferably comprises at least one biologically active part in a carbohydrate utilization-related or multidrug transporter protein. Within this fusion protein, the term “operably linked” means that a carbohydrate utilization-related or multidrug transporter polypeptide and a polypeptide that is not a carbohydrate utilization-related or multidrug transporter are in frame. It means that they are fused together. A polypeptide that is not a carbohydrate utilization-related or multidrug transporter may be fused to the N-terminus or the C-terminus of a carbohydrate utilization-related or multidrug transporter polypeptide.

  By expressing the linked coding sequence, two linked heteroamino acid sequences are obtained that form a fusion protein. A carrier sequence (polypeptide that is not a carbohydrate utilization-related or multidrug transporter) may encode a carrier polypeptide that enhances or increases expression of the fusion protein in a bacterial host. The portion of the fusion protein encoded by the carrier sequence (ie, carrier polypeptide) can be a protein fragment, an entire functional portion, or an entire protein sequence. The carrier region or polypeptide can also be designed to be used for purification of the fusion protein by an antibody or affinity purification specific for that carrier polypeptide. Similarly, the physical properties of the carrier polypeptide can be exploited to allow selective purification of the fusion protein.

  Specific carrier polypeptides include superoxide dismutase (SOD), maltose binding protein (MBP), glutathione-S-transferase (GST), N-terminal histidine (His) tag, and the like. Any carrier polypeptide that enhances the expression of a carbohydrate utilization-related protein or a multidrug transporter protein as a fusion protein can be used in the present invention, and is not limited to the carrier polypeptide described above.

  In one embodiment, the fusion protein is a fusion protein of GST and a carbohydrate utilization-related or multidrug transporter, in which the carbohydrate utilization-related or multidrug transporter sequence is fused to the C-terminus of the GST sequence is doing. In another embodiment, the fusion protein is a carbohydrate utilization related-immunoglobulin fusion protein, wherein all or part of the carbohydrate utilization related protein is fused to a sequence derived from a member of the immunoglobulin protein family. In other embodiments, the fusion protein includes a multidrug transporter protein of the invention. The carbohydrate utilization-related-immunoglobulin fusion protein or multidrug transporter-immunoglobulin fusion protein of the present invention is an immunogen for producing an anti-carbohydrate utilization-related antibody or anti-multidrug transporter-related antibody in the body of a subject. In addition, in order to purify a carbohydrate utilization-related ligand or a multidrug transporter-related ligand, and a carbohydrate utilization-related protein or a multidrug transporter protein and a carbohydrate utilization-related ligand or a multidrug transporter ligand It can be used in screening assays to identify molecules that inhibit the interaction.

One skilled in the art will appreciate that the specific carrier polypeptide is selected in view of the purification scheme. For example, His tags, GST, and maltose binding proteins are representative of carrier polypeptides that can be used with readily available affinity columns and bind to and elute such columns. Thus, if the carrier polypeptide is an N-terminal His tag, such as hexahistidine (His ₆ tag), a carbohydrate utilization-related or multidrug transporter fusion protein can be obtained from a metal chelate resin, such as nickel nitrilotriacetic acid (Ni- NTA), nickel iminodiacetic acid (Ni-IDA), and a cobalt-containing resin (Co resin). See, for example, Steinert et al. (1997) QIAGEN News 4: 11-15, which is incorporated herein by reference in its entirety. When the carrier polypeptide is GST, the carbohydrate utilization-related or multidrug transporter fusion protein can be purified using a matrix containing glutathione agarose beads (Sigma or Pharmacia Biotech). When is a maltose binding protein (MBP), a carbohydrate utilization-related or multidrug transporter fusion protein can be purified using a matrix comprising an agarose resin derivatized with amylose.

  The chimeric protein or fusion protein of the present invention is preferably prepared by standard recombinant DNA methods. For example, DNA fragments encoding different polypeptide sequences may be linked together in frame, and a fusion gene can be synthesized with an automatic DNA synthesizer or the like. Alternatively, a chimeric gene sequence can be prepared by PCR amplification, annealing, and further reamplification of a gene fragment using an anchor primer that provides a complementary overhang between two consecutive gene fragments (for example, Ausubel et al., Eds. (1995) Current Protocols in Molecular Biology (Greene Publishing and Wiley-Interscience, New York)). Furthermore, a nucleic acid encoding a carbohydrate utilization-related or multidrug transporter may be cloned into a commercially available expression vector and linked in-frame to an existing fusion moiety.

  Fusion protein expression vectors are usually designed to facilitate removal of the carrier polypeptide so that the carbohydrate utilization-related protein or multidrug transporter protein can retain its own natural biological activity. Methods for cleaving the fusion protein are known in the art. See, for example, Ausubel et al., Eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.). The fusion protein can be chemically cleaved using reagents such as cyanogen bromide, 2- (2-nitrophenylsulfenyl) -3-methyl-3'-bromoindolenine, hydroxylamine, or low pH reagents. Chemical cleavage is often performed under denaturing conditions in order to cleave proteins that otherwise become insoluble fusion proteins.

  When it is necessary to separate a carbohydrate utilization-related polypeptide or a multidrug transporter polypeptide from a carrier polypeptide and there is no intrinsic cleavage site between these fusion polypeptides, a specific protease cleavage site Can be included to design fusion constructs to facilitate enzymatic cleavage and removal of the carrier polypeptide. In this way, a linker sequence containing a coding sequence of a peptide having a cleavage site specific for a given enzyme is converted into a carrier polypeptide (eg, MBP, GST, SOD, or N-terminal His tag) coding sequence, It can be fused in-frame with the coding sequence of the quality utilization related polypeptide or multidrug transporter polypeptide. Suitable enzymes with specificity for the cleavage site include, but are not limited to, factor Xa, thrombin, enterokinase, remin, collagenase, and tobacco etch virus (TEV) protease. The cleavage sites for these enzymes are well known in the art. Thus, for example, when factor Xa is used to cleave a carrier polypeptide from a carbohydrate utilization-related polypeptide or a multidrug transporter polypeptide, it encodes a cleavage site sensitive to factor Xa, eg, a sequence called IEGR Fusion constructs can be designed to include a linker sequence (eg, Nagai and Thogersen (1984) Nature 309: 810-812; Nagai and Thogersen (1987) Meth. Enzymol, incorporated herein by reference). 153: 461-481; and Pryor and Leiting (1997) Protein Expr. Purif. 10 (3): 309-319). When thrombin is used to cleave a carrier polypeptide from a carbohydrate utilization related polypeptide or a multidrug transporter polypeptide, a linker sequence encoding a thrombin sensitive cleavage site, for example, the sequence LVPRGS or VIAGR, is used. Fusion constructs can be designed to include (eg, Pryor and Leiting (1997) Protein Expr. Purif. 10 (3): 309-319, and Hong et al. (1997) Chin. Med. Sci. J. 12 (3): 143-147; the disclosures of which are incorporated herein by reference). The cleavage sites for TEV protease are known in the art. See, for example, the cleavage site described in US Pat. No. 5,532,142, which is incorporated herein by reference in its entirety. See also the discussion section of Ausubel et al., Eds. (1998) Current Protocols in Molecular Biology (John Wiley & Sons, Inc.), Chapter 16.

(antibody)
The isolated polypeptide of the present invention can be used as an immunogen for producing an antibody that specifically binds to a carbohydrate utilization-related protein or a multidrug transporter protein, or for in vivo antibody production. . The full length of a carbohydrate utilization-related protein or multidrug transporter protein can be used as an immunogen, or using a peptide fragment of a carbohydrate utilization-related protein or multidrug transporter protein as described herein You can also. The antigenic peptide of the carbohydrate utilization-related protein or multidrug transporter protein has at least 8, preferably 10, 15, 20, or 30 amino acids from the amino acid sequence represented by an even sequence number among SEQ ID NOs: 1 to 320 It includes residues and includes epitopes so that antibodies produced against such peptides form specific immune complexes with carbohydrate utilization related proteins or multidrug transporter proteins.
A preferred epitope included in the antigenic peptide is a region of a carbohydrate utilization-related protein or a multidrug transporter protein, and a region present on the surface of the protein, for example, a hydrophilic region.

(Recombinant expression vectors and host cells)
The nucleic acid molecule of the present invention may be contained in a vector, preferably an expression vector. A “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. An expression vector contains one or more regulatory sequences and directs the expression of genes that are operably linked. “Operably linked” means that the nucleotide sequence of interest is capable of expression of the nucleotide sequence (eg, in an in vitro transcription / translation system or when the vector is introduced into a host cell). In a host cell) means linked to a regulatory sequence. The term “regulatory sequence” is intended to include other expression control elements such as regulatable transcription promoters, operators, enhancers, transcription terminators, and translational regulatory sequences (eg, Shine-Dalgarno). Consensus sequence, start codon, stop codon). These regulatory sequences vary depending on, for example, the host cell used.

  The vector may be replicated autonomously in the host cell (episomal vector) or may be integrated into the host cell genome and replicated with the host genome (non-episoap mammalian vector). An integrating vector usually contains at least one sequence homologous to the bacterial chromosome, thereby allowing recombination to occur between the homologous DNA present in the vector and the bacterial chromosome. Yes. The integrating vector may also contain bacteriophage or transposon sequences. Episomal vectors or plasmids are circular double stranded DNA loops into which additional DNA segments can be ligated. When using recombinant DNA methods, plasmids that can be stably maintained in the host are usually the preferred form of expression vectors.

  An expression construct or vector encompassed by the present invention includes a nucleic acid construct of the present invention in a form suitable for expression of the nucleic acid in a cell. Expression in prokaryotic and plant cells is also encompassed by the present invention. Those skilled in the art will appreciate that the design of the expression vector will depend on factors such as the choice of the host cell to be transformed and the level of expression of the desired protein. By introducing the expression vector of the present invention into cells, proteins or peptides encoded by the nucleic acids described herein, including fusion proteins or peptides (eg, carbohydrate utilization related proteins or multidrug transporter proteins) , Carbohydrate utilization-related proteins or multidrug transporter protein variants, fusion proteins, etc.).

(Bacterial expression vector)
Regulatory sequences include those that direct constitutive expression of nucleotide sequences and those that direct nucleotide sequence expression induced only under certain environmental conditions. Any DNA sequence capable of binding bacterial RNA polymerase and initiating downstream (3 ′) transcription of a coding sequence (eg, a structural gene) into mRNA is a bacterial promoter. A promoter has a transcription initiation region, which is usually located near the 5 ′ end of the coding sequence. This transcription initiation region usually includes an RNA polymerase binding site and a transcription initiation site. Bacterial promoters may also have a second domain called the operator. The operator may overlap with the RNA polymerase binding site that is adjacent and the site where RNA synthesis begins. Operators allow for negatively regulated (inducible) transcription because gene repressor proteins can bind to the operator and thereby inhibit transcription of certain genes. Constitutive expression may occur in the absence of negative regulatory elements such as operators. In addition, gene activation protein binding sequences may result in positive regulation. This sequence, if present, is usually near (5 ') the RNA polymerase binding sequence.

  An example of a gene activating protein is the catabolite gene activating protein (CAP), which assists in the initiation of transcription of the Escherichia coli lac operon (Raibaud et al. (1984) Annu. Rev. Genet 18: 173). Thus, regulatory expression can be positive or negative, which promotes or represses transcription. Other examples of positive and negative regulatory elements are well known in the art. Various promoters that can be included in the protein expression system include, but are not limited to, the T7 / LacO hybrid promoter, the trp promoter, the T7 promoter, the lactose promoter, and the bacteriophage λ promoter. Any suitable promoter can be used in the practice of the present invention, including native or heterologous promoters. The heterologous promoter may be constitutively active or inducible. Non-limiting examples of heterologous promoters are shown in US Pat. No. 6,242,194.

  Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences for sugar metabolizing enzymes such as galactose, lactose (lac) (Chang et al. (1987) Nature 198: 1056), and maltose. Further, a promoter sequence derived from a biosynthetic enzyme (trp) such as tryptophan (Goeddel et al. (1980) Nucleic Acids Res. 8: 4057; Yelverton et al. (1981) Nucleic Acids Res. 9: 731; 4738921; European Patent Publication Nos. 367776 and 121775). Beta-lactamase (bla) promoter system (Weissmann, (1981) "The Cloning of Interferon and Other Mistakes," in Interferon 3 (ed. I. Gresser)); bacteriophage lambda PL (Shimatake et al: (1981) Nature 292: 128); the arabinose-inducible araB promoter (US Pat. No. 5,028,530); and the T5 (US Pat. No. 4,689,406) promoter system also provide useful promoter sequences. See also Balbas (2001) Mal. Biotech. 19: 251-267, which discusses the E. coli expression system.

  Furthermore, synthetic promoters that do not exist in nature also function as bacterial promoters. For example, the transcriptional activation sequence of one bacterial promoter or bacteriophage promoter can be linked to the operon sequence of another bacterial promoter or bacteriophage promoter to generate a synthetic hybrid promoter (US Pat. No. 4,551,433). For example, the tac promoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21) and the trc promoter (Brosius et al. (1985) J Biol. Chem. 260: 3539-3541) is a trp-lac hybrid promoter composed of both a trp promoter and a lac operon sequence regulated by a rack repressor. The tac promoter further has a function as an inducible regulatory sequence. Thus, expression of a coding sequence operably linked to the tac promoter can be induced, for example, by cell culture with the addition of isopropyl-1-thio-β-D-galactoside (IPTG). In addition, natural promoters other than bacteria that have the ability to bind bacterial RNA polymerase and initiate transcription are also included in bacterial promoters. Natural promoters from non-bacteria can also be conjugated with a compatible RNA polymerase so that any prokaryotic gene is expressed at high levels. The bacteriophage T7 RNA polymerase / promoter system is an example of a coupled promoter system (Stadier et al. (1986) J. Mol. Biol. 189: 113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82. : 1074). In addition, the hybrid promoter may consist of a bacteriophage promoter and an E. coli operator region (European Patent No. 267851).

  The vector may further have a gene encoding a repressor (or inducer) of the promoter. For example, the inducible vector of the present invention regulates transcription from a Lac operator (LacO) by expressing a gene encoding a LacI repressor protein. Other examples include the use of the lexA gene to regulate pRecA expression and the use of trpO to regulate ptrp. Alleles of these genes that enhance the degree of repression (eg, lacIq) or modify the mode of induction (eg, λCI857 that gives λpL heat-inducibility, or λCI + that gives λpL chemical inductivity) Can also be used.

  In addition to a functional promoter sequence, an efficient ribosome binding site is also useful for expression of the fusion construct. In prokaryotes, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes a start codon (ATG) and a sequence of 3-9 nucleotides in length 3-11 nucleotides upstream of the start codon (Shine et al. (1975) Nature 254: 34). The SD sequence is believed to promote the binding of mRNA to the ribosome by base pairing between the SD sequence and the 3 ′ end of bacterial 16S ribosomal RNA (Steitz et al. (1979) “Genetic Signals and Nucleotide”. Sequences in Messenger RNA, "in Biological Regulation and Development: Gene Expression (ed. RF Goldberger, Plenum Press, NY).

  Glucose utilization related proteins can also be secreted from cells by creating a chimeric DNA molecule that encodes a protein containing a signal peptide sequence fragment that results in carbohydrate utilization related and multidrug transporter polypeptide secretion in bacteria. (U.S. Pat. No. 4,336,336). The signal sequence fragment usually directs protein secretion from the cell and encodes a signal peptide composed of hydrophobic amino acids. Proteins are secreted either in the growth medium (Gram positive bacteria) or in the periplasmic space located between the inner and outer membranes of the cells (Gram negative bacteria). There is preferably a processing site encoded between the signal peptide fragment and the carbohydrate utilization-related protein or multidrug transporter protein that can be cleaved either in vivo or in vitro.

  The DNA encoding the appropriate signal sequence is the E. coli outer membrane protein gene (ompA) (Masui et al. (1983) FEBS Lett. 151 (1): 159-I64; Ghrayeb et al. (1984). ) EMBOJ 3: 2437-2442), and E. coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985) Proc. Natl. Acad Sci. 82: 7212). Can be obtained from Other prokaryotic signals include, for example, a signal sequence from penicillinase Ipp; or a thermostable enterotoxin II leader sequence.

  Bacteria such as L. acidophilus typically utilize the start codon ATG, which specifies the amino acid methionine (which is altered to N-formylmethionine in prokaryotes). Bacteria also recognize other initiation codons, such as the codons GTG and TTG, which encode valine and leucine, respectively. However, when these codons are used as initiation codons, they direct the incorporation of methionine rather than the amino acid normally encoded by these codons. Lactobacillus acidophilus NCFM recognizes such another start site and incorporates methionine as the first amino acid.

  Usually, the transcription termination sequence recognized by bacteria is a regulatory region located 3 'to the translation stop codon, and thus sandwiches the coding sequence together with the promoter. These sequences direct the transcription of mRNA that can be translated into a polypeptide encoded by the DNA. Transcription termination sequences often include DNA sequences (about 50 nucleotides) that can form a stem loop structure that assists in terminating transcription. Examples of these include transcription termination sequences derived from genes with strong promoters such as the E. coli trp gene and other biosynthetic genes.

  The expression vector has a plurality of restriction sites to be inserted into a carbohydrate utilization-related sequence or a multidrug transporter sequence in order to undergo transcriptional regulation of the regulatory region. A selectable marker gene to ensure that the vector is maintained in the cell can also be included in the expression vector. Preferred selective markers include those that confer drug resistance such as ampicillin, chloramphenicol, erythromycin, kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev. Microbial. 32: 469). Selective markers may allow cells to grow on minimal media or in the presence of toxic metabolites, including biosynthetic genes such as genes for the histidine, tryptophan and leucine biosynthetic pathways. Is included.

  The regulatory region may be native (homo) or foreign (heterologous) to the cells and / or nucleotide sequences of the present invention. The regulatory region may be natural or synthesized. That the region is “foreign” or “heterologous” for the cell means that the cell into which the region is introduced originally does not have such a region. That the region is “foreign”, ie, “heterologous” to the carbohydrate utilization-related nucleotide sequence or multidrug transporter nucleotide sequence of the present invention means that the carbohydrate utilization-related nucleotide sequence of the present invention is operably linked. Or for multidrug transporter nucleotide sequences, it means that the region is not a native or naturally occurring region. Such a region may be derived, for example, from a phage. Such sequences are preferably expressed using heterologous regulatory regions, but native regions can also be used. In some cases, such constructs are also expected to change the intracellular expression level of carbohydrate utilization-related proteins or multidrug transporter proteins. Thus, the cell phenotype may change.

  In preparing the expression cassette, various DNA fragments may be manipulated to provide the DNA sequence in an appropriate orientation and, if necessary, in an appropriate reading frame. For this purpose, adapters or linkers may be used to link DNA fragments, and other operations are performed for convenient restriction sites, unnecessary DNA removal, restriction site removal, etc. There is also. To do these things, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (eg transfer and conversion) may be performed.

  The present invention further provides a recombinant expression vector comprising in the antisense direction an expression vector in which the DNA molecule of the present invention has been cloned. That is, the DNA molecule of the present invention is operably linked to a regulatory sequence so that expression of an RNA molecule that is antisense to the carbohydrate utilization-related or multidrug transporter mRNA occurs (by transcription of the DNA molecule). . Regulatory sequences operably linked to the nucleic acid cloned in the antisense orientation can be selected to cause continuous or inducible expression of the antisense RNA molecule. The antisense expression vector may be a recombinant plasmid or a recombinant phagemid, in which an antisense nucleic acid is produced under the control of a highly efficient regulatory region and the activity of this regulatory region. Is determined by the cell type into which the vector has been introduced. For a discussion on the regulation of gene expression using antisense genes, see Weintraub et al. (1986) Reviews Trends in Genetics, Vol. 1 (1).

  Alternatively, some of the above components can be included in the transformation vector. Transformation vectors are usually composed of selectable markers and are maintained by replication units or developed into integrating vectors as described above.

(Plant expression vector)
For expression in plant cells, the expression cassette has a transcription initiation region operably linked to the nucleotide sequence of the present invention. Such expression vectors include various restriction sites to allow insertion of nucleotide sequences under transcriptional regulation in the regulatory region. Furthermore, the expression cassette may contain a selection marker such as a gene that provides herbicidal resistance or antibacterial resistance such as tetracycline resistance, hygromycin resistance, ampicillin resistance, glyphosate resistance and the like.

  The expression cassette contains a transcription initiation region and translation initiation region, a nucleotide sequence of the present invention, and a transcription termination region and translation termination region (stop region) that function in plants in the transcription direction from 5 ′ to 3 ′. . The stop region may be derived from a transcription initiation region containing a promoter nucleotide sequence, may be derived from the nucleotide sequence of the present invention, or may be derived from another. Suitable stop regions in the art are known and exemplified by A. tumefaciens Ti-plasmid stop regions, such as the octopine synthase and nopaline synthase stop regions. It is not limited to. Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. 1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic See also Acid Res. 15: 9627-9639.

  The expression cassette containing the nucleotide sequence of the present invention may further contain at least one nucleotide sequence of a gene that cotransforms an organism. Alternatively, such additional sequences may be provided in a separate expression cassette.

  The expression cassette may include a 5 'non-translated leader sequence or a 5' non-coding sequence. As used herein, “5 ′ leader sequence”, “translation leader sequence”, and “5 ′ non-coding sequence” are transcribed into RNA and completely processed upstream (5 ′ side) of the translation initiation codon. It refers to the DNA sequence portion of the gene between the promoter and coding sequence that is present in the mRNA. 5 'untranslated leader sequences are typically characterized as part of an mRNA molecule that normally extends from a 5' cap (CAP) site to the AUG protein translation initiation codon. Translation leader sequences can also affect the processing of primary transcripts into mRNA, mRNA stability, and translation efficiency (Turner et al. (1995) Molecular Biotechnology 3: 225). Thus, the translation leader sequence plays an important role in the regulation of gene expression. Translation leaders are known in the art and include picornavirus leaders such as the EMCV leader (encephalomyocarditis 5 'non-coding region) (Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA 86: 6126-6130); potyvirus leaders such as TEV leader (Etch Virus virus (Allison et al. (1986) Virology 154: 9-20)); MDMV leader (sugarcane mosaic virus) (Maize Dwarf Mosaic Virus)); human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leader derived from alfalfa mosaic virus coat protein mRNA (AMVRNA4) (Jobling et al. (1987) Nature 325: 622-625); Tobacco Mosaic Virus Reader (TMV) (Gallie et al. (1989) Molecular Biology of RNA, pages 237-256); Sorghum chloro tick Mottle Virus (maize chlorotic mottle virus leader (MCMV)) (Lommel et al (1991) Virology 81:. 382-385) and is exemplified, but not limited to.

  Other methods known to enhance translation and / or mRNA stability can also be utilized. Other methods include, for example, maize ubiquitin intron (Christensen and Quail (1996) Transgenic Res. 5: 213-218 and Christensen et al. (1992) Plant Molecular Biology 18: 675-689) or maize AdhI intron (Kyozuka et al. al. (1991) Mol. Gen. Genet. 228: 40-48 and Kyozuka et al. (1990) Maydica 35: 353-357). A number of intron sequences have been shown to enhance expression, particularly in monocotyledonous cells. The maize AdhI gene intron is known to significantly enhance wild-type gene expression in the presence of homologous promoters when introduced into maize cells. Intron 1 is particularly effective and is known to enhance expression in fusion constructs with the chloramphenicol acetyltransferase gene (Callis et al. (1987) Genes Develop. 1: 1183-1200). In the same experimental system, the maize bronze 1 gene showed similar effects on enhanced expression. The AdhI intron has been shown to enhance CAT expression 12-fold (Mascarenhas et al. (1990) Plant Mol. Biol. 6: 913-920). Intron sequences are most commonly incorporated in plant transformation vectors, usually in their untranslated leaders.

  The expression cassette having the promoter sequence of the present invention may further contain a 3 'non-coding sequence. “3 ′ non-coding sequence” or “3 ′ untranslated region” is located 3 ′ (downstream) of the coding sequence, and is used to add a polyadenylation signal sequence and a polyadenylate tail at the 3 ′ end of the mRNA precursor. By nucleotide sequence is meant including other sequences encoding regulatory signals that can be affected. The 3 'untranslated region includes a region in mRNA, usually a region that begins with a translation termination codon and extends at least beyond the polyadenylation site. It is known that the untranslated sequence located at the 3 'end of the gene affects the expression level of the gene. Ingelbrecht et al. (See Plant Cell, 1: 671-680, 1989) evaluated the importance of these elements and found that expression in stable plants varies greatly depending on the origin of the 3 'untranslated region. It was. Highest level expression by using 3 'untranslated region in association with octopine synthase, Arabidopsis 2S seed protein, Arabidopsis rbcS small subunit, carrot extension, and Antirrhinium chalcone synthase There was a 60-fold difference between the constructs that showed the expression (constructs containing the 3 ′ untranslated region of rbcS) and the constructs that showed the lowest level of expression (constructs containing the 3 ′ region of chalcone synthase). Yes.

  Transcription levels are also enhanced by using enhancers in combination with the promoter regions of the present invention. An enhancer is a nucleotide sequence that has the effect of enhancing the expression of a promoter region. Enhancers are known in the art and include the SV40 enhancer region, 35S enhancer element, and the like.

  In preparing an expression cassette, various DNA fragments are manipulated to provide the correct orientation and, if necessary, the correct reading frame DNA sequence. Adapters or linkers may be used to join DNA fragments together, and other manipulations may be performed to provide suitable restriction sites. Restriction sites may be added or removed, excess DNA may be removed, or other modifications may be made to the sequences of the invention. Therefore, in vitro mutagenesis, primer repair, restriction, annealing, for example, re-substitution such as transfer and base conversion may be performed.

  In addition to the selectable markers that provide antibacterial and herbicidal resistance as described above, other genes that are useful for recovery from transgenic events but are unnecessary for the final product include GUS (b-glucoronidase; Jefferson (1987) Plant Mol. Biol. Rep. 5: 387), GFP (green florescence protein; Chalfie et al. (1994) Science 263: 802), luciferase (Riggs et al. (1987) Nucleic Acids Res. 15 (19): 8115 and Luehrsen et al. (1992) Methods Enzymol. 216: 397-414), and maize genes encoding anthocyanin production (Ludwig et al. (1990) Science 247: 449), but are not limited to these.

  The nucleic acids of the present invention are useful in methods for expressing nucleotide sequences in plants. This is achieved by transforming a target plant cell with an expression cassette comprising a promoter operably linked to the nucleotide sequence described herein and regenerating a stably transformed plant from the plant cell. The An expression cassette comprising a promoter sequence operably linked to the nucleotide sequence of the present invention can be used for transformation of any plant. In this way, genetically modified, ie transgenic or transformed plants, plant cells, plant tissues, seeds, roots etc. can be obtained.

(Microbial cells or bacterial cells)
Production of bacteria having heterologous phage resistance genes, preparation of starter cultures of such bacteria, and substrate fermentation (especially food substrates such as milk) can be performed by known methods.

  “Introducing” a nucleic acid means introduction into a prokaryotic or eukaryotic cell by conventional transformation or transfection techniques, or by phage-mediated infection. As used herein, the terms “transformation”, “transduction”, “conjugation” and “protoplast fusion” refer to exogenous nucleic acids (eg, DNA) in cells. Refers to various techniques recognized in the art for introduction into, such as coprecipitation with calcium phosphate or calcium chloride, DEAE-dextran transfection, lipofection or electroporation. Suitable methods for transforming and transfecting cells include Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York) and others. In the laboratory manual. “Introducing” a polypeptide or microorganism of the invention means introducing the polypeptide or microorganism into the host by ingestion, topical application, nasal cavity, suppository, genitourinary or oral application.

  Bacterial cells used for the production of the carbohydrate utilization-related polypeptide or multidrug transporter polypeptide of the present invention include Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, Incubate in an appropriate medium as described in New York.

  Bacterial strains encompassed by the present invention include pure biological cultures of bacteria comprising at least one nucleotide or amino acid sequence of the present invention. Such bacterial strains include: the ability to transport carbohydrates into and out of cells as compared to wild-type Lactobacillus acidophilus, and this altered ability is Lactobacillus acidophilus obtained by expressing at least one of the peptides; the ability to accumulate carbohydrates is modified compared to wild-type Lactobacillus acidophilus, and the altered ability is the carbohydrate utilization type of the present invention Lactobacillus acidophilus obtained by expressing at least one of the polypeptides; the ability to use carbohydrates as an energy source is modified as compared to wild-type Lactobacillus acidophilus. Lactoba obtained by expressing at least one of carbohydrate-utilizing polypeptides Luz acidophilus; providing a food whose flavor is modified by fermentation compared to wild-type Lactobacillus acidophilus, and the modified flavor is obtained by expressing at least one of the carbohydrate-based polypeptides of the present invention Lactobacillus acidophilus produced; a food having a texture modified by fermentation compared to wild-type Lactobacillus acidophilus, wherein the modified texture expresses at least one of the carbohydrate-based polypeptides of the present invention Compared to wild-type Lactobacillus acidophilus, the ability to produce carbohydrates is modified, and such ability is obtained by expressing at least one of the carbohydrate-utilizing polypeptides of the present invention. Lactobacillus acidophilus; wild type Compared to Bacillus acidophilus, the ability to survive under food processing and storage conditions is modified, and such modified ability is obtained by expressing at least one of the carbohydrate-based polypeptides of the present invention. Acidophilus; the ability to survive in the gastrointestinal tract is modified compared to wild-type Lactobacillus acidophilus, and the modified ability is obtained by expressing at least one of the carbohydrate-based polypeptides of the present invention. Bacillus acidophilus; compared to wild-type Lactobacillus acidophilus, viability when contacted with an antimicrobial polypeptide or toxin is altered, such altered ability being at least one of the multidrug transporter polypeptides of the invention Lactobacillus acidophilus obtained by expressing Luz.

(Transgenic plants and plant cells)
As used herein, “transformed plant” and “transgenic plant” mean a plant having a heterologous polynucleotide in its genome. Heterologous polynucleotides are generally stably integrated into the genome of the transgenic or transformed plant so that such heterologous polypeptides are passaged from generation to generation. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. As used herein, the term “transgenic” includes any cell, cell line, callus, tissue, plant part, or plant whose genotype is altered by the presence of a heterologous nucleic acid, from the beginning. It is to be understood that this variation also includes those produced from the initial transgenic by sexual or asexual reproduction. As used herein, the term “transgenic” includes genomic (chromosomal or extrachromosomal) changes by conventional plant growth methods, naturally occurring events such as random cross-pollination, non-recombinant viral infections. It does not include bacterial transformation without recombination, transposition without recombination, and genomic changes due to natural mutation.

  A transgenic “event” is a transformation of a plant cell with a heterologous DNA construct containing a nucleic acid expression cassette containing the desired transgene and inserting the transgene into the plant genome to regenerate and identify the plant population. This is done by selecting a specific plant that is characterized by insertion into the genomic location. Events are characterized by phenotype by transgene expression. The event is a genetic make-up of the plant at the genetic level. The term “event” also refers to a progeny plant produced by sexual outcrossing between a transformant and another species having heterologous DNA.

  The term “plant” as used herein includes whole plants, plant organs (leaves, stems, roots, etc.), seeds, plant cells, and progeny plants. A part of the transgenic plant included in the scope of the present invention originates from a transgenic plant transformed with the DNA molecule of the present invention or a progeny plant thereof, and thus at least a part thereof contains a transgenic cell. It should be understood that plant cells, protoplasts, tissues, callus, germs are included as well as flowers, pollen, cocoons, stems, fruits, ovules, leaves, roots.

  As used herein, the term “plant cell” includes seed suspension cultures, embryos, division regions, callus tissue, leaves, roots, buds, gametophytes, spores, pollen, microspores. It is not limited to. The classification of plants that can be used in the method of the present invention generally includes a broad range of classifications of higher plants that can be transformed, and includes both monocotyledonous plants and dicotyledonous plants.

  The present invention is used for transformation in any plant species, including but not limited to monocotyledonous and dicotyledonous plants. Target plants include corn (Zea mays), rape seeds (eg, B. napus, B. rapa, B. juncea), especially rape seeds that are useful as seed oils, alfalfa ( Medicago sativa, rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet millet (eg; Pennisetum glaucum), millet (Panicum miliaceum), millet (Setaria italica) ), Millet (Eleusine coracana)), acid flower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanut (Arachis) hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), Coconut (Cocos nucifera), pineapple (Ananas comosus), citrus (Citrus spp.), Cocoa (Theobroma cacao) tea (Camellia sinensis), banana (Musa spp.), Avocado (Persea americana)), fig (Ficus casica), Gaba (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris) Examples include, but are not limited to, sugar cane related species (Saccharum spp.), Oats, barley, vegetables, foliage plants, and conifers.

  Vegetables include tomatoes (Lycopersicon esculentum), lettuce (eg Lactuca sativa), green beans (green beans (Phaseolus vulgaris)), laima beans (Phaseolus limensis), peas (Lathyrus spp.) ), And cucumber vegetables such as C. sativus, C. cantalupensis, and muskmelon (C. melo). Houseplants include azalea (Rhododendron spp.), Hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), rose (Rosa spp.), Tulip (Tulipa spp.), Narcissus (Narcissus spp.), Petunia hybrida , Carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima) and chrysanthemum. Conifers used in practicing the present invention include, for example, Pinus taeda, slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodge pine (Pinus contorta) and montery pine (Pinus radiata). Pine etc .; Bay pine (Pseudotsuga menziesii); American tsuka (Tsuga canadensis); Bee spruce (Picea glauca); Redwood (Sequoia sempervirens); European fir (Abies amabilis) and fir such as Balsam fir (Abies balsamea); and Western red Sugi such as cedar (Thuja plicata) and Alaska yellow cedar (Chamaecyparis nootkatensis) are included.

  The method of the present invention does not depend on a specific method for introducing a nucleotide construct into a plant, but may be any method as long as the nucleotide construct reaches the inside of at least one cell of the plant. Methods for introducing nucleotide constructs into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, viral methods, and the like.

  “Transient transformation method” means that a nucleotide construct introduced into a plant is not integrated into the genome of the plant. “Stable transformation” means that a nucleotide construct introduced into a plant can be integrated and passaged into the genome of the plant. “Primary transformants” and “T0 generation” transgenic plants are of the same genetic generation as the originally transformed tissue (ie, have not undergone meiosis or pollination since transformation). “Secondary transformants” and “T1, T2, T3 and later generations” are transgenic plants that have undergone one or more meiosis or pollination cycles from the primary transformant, It may be derived from self-pollination of the primary or secondary transformant, or the primary or secondary transformant may be obtained by cross-pollinating with another transformed plant or a non-transformed plant.

  Transformation protocols and protocols for introducing nucleotide sequences into plants vary depending on the plant or plant cell type that is the target of transformation, ie, monocotyledonous or dicotyledonous. The nucleotide constructs of the present invention may be introduced into plants by any of the methods known in the art and include methods for contacting plants with viruses or viral nucleic acids (US Pat. Nos. 5,889,191, 5889190, incorporated herein by reference). No. 5866785, 5589367 and 5316931), microinjection method (Crossway et al. (1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606), Agrobacterium-mediated transformation methods (US Pat. Nos. 5,981,840 and 5,563,055), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722) , As well as ballistic particle acceleration methods (e.g. U.S. Pat. Nos. 4,945,050, 5879918, 588624) Includes item and see No. 5932782), these are incorporated herein by reference in its entirety, it is not limited to these methods.

  Transformed cells are grown on plants by methods known in the art. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. Such plants are then grown and the same transformant or another strain is pollinated to identify hybrids that express the desired phenotypic characteristics. To confirm that the expression of the desired phenotypic characteristics is stably maintained and passaged, grow more than 2 generations and harvest the seeds to see if the desired phenotypic characteristics are obtained. Good. Thus, as used herein, “transformed seed” means a seed having a nucleotide construct that is stably integrated into the plant genome.

(how to use)
Methods for altering the expression of carbohydrate utilization-related or multidrug transgenic genes or proteins in an organism are provided. In one embodiment, a microbial strain is provided that can modify the properties of the microorganism used for fermentation to utilize alternative carbohydrates as an energy or carbon source. Such modifications can provide a new ability to synthesize, transport, accumulate and degrade carbohydrates. Alternatively, such modifications can acquire the ability to survive contact with antimicrobial polypeptides such as antibiotics and toxins. These new capabilities can also increase the viability of microorganisms, for example, in the gastrointestinal tract and under stress conditions in food processing and storage, thereby increasing the usefulness of the microorganisms in the fermentation of various foods. Probiotic activity persists. These new capabilities also allow microorganisms to bring various flavors and textures to the product after fermentation. Furthermore, these new capabilities allow bacteria to produce modified carbohydrates, exopolysaccharides, or cell surface polysaccharides. In another embodiment, the plant characteristics are modified to provide similar capabilities. Such ability is provided by the nucleotide and amino acid sequences disclosed in the present invention.

  Generally, the above-described method is a method including introducing or overexpressing one or more proteins involved in carbohydrate utilization or multidrug resistance. “Introducing” means that the target protein is expressed in a modified cell when it is not expressed in an unmodified cell. The term “overexpressed” means that the expression level of the target protein is increased in the modified organism compared to the production amount of the target protein in the unmodified wild-type organism. In particular, homofermentable lactic acid bacteria are relatively simple in their metabolism and have little overlap between energy and biosynthetic metabolism, making them ideal targets for metabolic engineering ((Hugenholz and Kleerebezem ( 1999) Current Opin.Biotech.10: 492-497) Expression of bacterial genes in plants is known in the art, for example, Shewmaker et al. (1994) Plant Physiol.104: 1159-1166; (1997) Plant Physiol. 113: 1177-1183; Blaszczyk et al. (1999) Plant J. 20: 237-243.

  Expression of one or more carbohydrate utilization-related proteins or multidrug transporter proteins alters the ability to transport carbohydrates or antimicrobial polypeptides, such as transporting bacteriocin into and out of cells.

  Dehydrogenase is also included as a carbohydrate utilization-related protein. Aldehyde dehydrogenase (EC: 1.2.1.3 and EC: 1.2.1.5) (PFAM Accession No. PF00171) uses a number of aliphatic and aromatic aldehydes using NADP as a cofactor. It is an enzyme that oxidizes. In mammals, at least four different forms of enzyme are known (Hempel et al. (1989) Biochemistry 28: 1160-7), which are class 1 (or Ald C) tetrameric cytoplasmic enzymes, Class 2 (or Ald M) tetrameric mitochondrial enzymes, class 3 (or Ald D) dimeric cytoplasmic enzymes, and class 4 microsomal enzymes. Aldehyde dehydrogenase has also been sequenced from fungal and bacterial species. A number of enzymes are known to be evolutionarily related to aldehyde dehydrogenases. Glutamate and cysteine residues have been implicated in the catalytic activity of aldehyde dehydrogenase in mammals. These residues are conserved in all enzymes of this family. The aldehyde dehydrogenase protein of the present invention includes the protein of SEQ ID NO: 228. The D-isomer specific 2-hydroxy acid dehydrogenase, the catalytic domain (PFAM accession number PF00389) protein of the present invention includes the protein of SEQ ID NO: 242. The D-isomer specific 2-hydroxy acid dehydrogenase, NAD binding domain (PFAM Accession No. PF02826) protein of the present invention includes the protein of SEQ ID NO: 242. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolysis by reversibly catalyzing the oxidation and phosphorylation of D-glyceraldehyde-3-phosphate to 1,3-diphospho-glycerate. And plays an important role in gluconeogenesis (Huang et al. (1989) J. Mol. Biol. 206: 411-24). This enzyme is a tetramer composed of the same subunits, each subunit having two conserved functional domains, an NAD binding domain and a highly conserved catalytic domain. SEQ ID NO: 248 has a glyceraldehyde 3-phosphate dehydrogenase, C-terminal domain (PFAM accession number PF02800) and a glyceraldehyde 3-phosphate dehydrogenase, NAD binding domain (PFAM accession number PF00044). L-Lactate dehydrogenase is a metabolic enzyme that catalyzes the conversion of L-lactic acid to pyruvate, which is the final step in anaerobic glycolysis. Maleate dehydrogenase catalyzes the interconversion of maleate and oxaloacetate. This enzyme is involved in the citrate cycle. The lactate / maleate dehydrogenase, α / β-C-terminal domain (PFAM accession number PF02866) protein and the lactate / maleate dehydrogenase, NAD binding domain (PFAM accession number PF00056) protein of the present invention have SEQ ID NOs: 222 and 246. Contains protein. Methods for measuring dehydrogenase activity are known in the art (eg, Ercolani et al. (1988) J. Biol. Chem. 263: 15335-41).

  Carbohydrate utilization related proteins also include O-glycosyl hydrolases. O-glycosyl hydrolase (EC 3.2.1.-) is a well-known enzyme that hydrolyzes glycosidic bonds between two or more carbohydrates, or between carbohydrate and non-saccharide components. Assays for measuring hydrolase activity are known in the art (eg, Avigad and Bauer (1966) Methods Enzymol. 8: 621-628; Neumann and Lampen (1967) Biochemistry 6: 468-475; Henry and Darbyshire ( 1980) See Phytochemistry 19: 1017-1020).

  α-Amylase catalytic domain proteins (PFAM accession number PF00128) are classified into family 13 of glycosyl hydrolases. The α-amylase and catalytic domain proteins of the present invention include the proteins of SEQ ID NOs: 108, 196, 240, 292 and 332.

  The β-galactosidase family (PFAM accession number PF02449) belongs to the glycosyl hydrolase 42 family. This enzyme catalyzes a terminal, non-reducing terminal β-D-galactosidase residue. The β-galactosidase protein of the present invention includes the protein of SEQ ID NO: 210. The β-galactosidase short C-terminal domain (PFAM accession number PF02930) is found at the carboxy-terminal part of the β-galactosidase (EC: 3.2.1.23) dimer short chain. The N-terminal domain of the short β-galactosidase chain (PFAM accession number PF02929) is found at the amino terminal end of the short β-galactosidase (EC: 3.2.1.23) dimer. These domains are also found in single-stranded β-galactosidase. The β-galactosidase short chain C-terminal protein of the present invention includes the protein of SEQ ID NO: 214, and the N-terminal domain protein of the present invention includes the protein of SEQ ID NO: 214.

  Glycoside hydrolase family 1 (PFAM accession number PF00232) includes β-glucosidase (EC: 3.2.1.21), β-galactosidase (EC: 3.2.1.23), 6-phospho-β- Galactosidase (EC: 3.2.1.85), 6-phospho-β-glucosidase (EC: 3.2.1.86), lactase-phlorizin hydrolase (EC: 3.2.1.62) (EC: 3.2.1.108), β-mannosidase (EC: 3.2.1.25), myrosinase (EC: 3.2.1.147), and other enzymes having various known activities are included. The glycoside hydrolase family 1 proteins of the present invention include the proteins of SEQ ID NOs: 10 and 220.

Glycoside hydrolase family 2 includes β-galactosidase (EC: 3.2.1.23), β-mannosidase (EC: 3.2.1.25), β-glucuronidase (EC: 3.2.1.31). Etc.), which have several known activities. These enzymes are E. coli lacZ (
1162279228773_1
lacZ) has a glutamic acid conserved residue that has been shown to be a normal acidic / basic catalyst at the enzyme active site (Gebler et al. (1992) J. Biol. Chem. 267: 11126-30). The glycosyl hydrolase family 2 immunoglobulin-like β-sandwich domain (PFAM accession number PF00703) represents the immunoglobulin-like β-sandwich domain. The sugar binding domain (PFAM accession number PF02837) has a jelly-roll fold. E. coli β-galactosidase has a TIM barrel-like core surrounded by four other large β-domains (PFAM accession number PF02836). SEQ ID NO: 212 includes these domains.

  Glycoside hydrolase family 31 (PFAM accession number PF01055) includes α-glucosidase (EC: 3.2.1.20), α-galactosidase (EC: 3.2.1.22), glucoamylase (EC: 3 2.1.3), sucrase-isomaltase (EC: 3.2.1.48) (EC: 3.2.1.10), α-xylosidase (EC: 3.2.1), and α -Enzymes with some known activity are included such as glucan lyase (EC: 4.2.2.13). In glycoside hydrolase family 31, a number of glycosyl hydrolases are grouped based on sequence similarity (Henrissat (1991) Biochem. J. 280: 309-16; Naim et al. (1991) FEBS Lett. 294: 109). -12). Aspartate has been implicated in the catalytic activity of sucrase, isomaltase, and lysosomal α-glucosidase (Hermans et al. (1991) J. Biol. Chem. 266: 13507-12). The glycoside hydrolase family 31 proteins of the present invention include the protein of SEQ ID NO: 226.

  Glycoside hydrolase family 32 (PFAM accession number PF00251) includes invertase (EC: 3.2.1.26), inulinase (EC: 3.2.1.7), levanase (EC: 3.2.1. 65), exoinulinase (EC: 3.2.1.80), sucrose: sucrose 1-fructosyltransferase (EC: 2.4.4.19), and fructan: fructan 1-fructosyltransferase (EC). : 2.4.1.100) and some other enzymes with known activity are included. The glycoside hydrolase family 32 proteins of the present invention include the proteins of SEQ ID NOs: 46 and 100.

  Enzymes with an isoamylase N-terminal domain (PFAM accession number PF02922) belong to the family 13 of glycosyl hydrolases. This domain is found in enzymes that act on branched substrates, namely isoamylases, pullulanases, and branching enzymes. Isoamylase hydrolyzes 1,6-α-D-glucoside branch bonds in glycogen, amylopectin and dextrin, and 1,4-α-glucan branch enzyme forms glycogen 1,6-glycoside bonds. The pullulanase debranches the starch. The isoamylase N-terminal domain protein of the present invention includes the protein of SEQ ID NO: 240.

  α-Galactosidase (EC: 3.2.1.22) (melibiase) (PFAM accession number PF02065) catalyzes the hydrolysis of melibiose to galactose and glucose (Dey and Pridham (1972) Adv. Enzymol. Relat. Areas Mol. Biol. 36: 91-130). α-Galactosidase exists in various organisms. There is a considerable degree of similarity in the sequences of various eukaryotic α-galactosidases. E. coli α-galactosidase (melA gene) requires NAD and magnesium as cofactors, but is not structurally related to eukaryotic enzymes. In contrast, α-galactosidase (rafA gene) encoded by an E. coli plasmid has a region of about 50 amino acids similar to the domain of α-galactosidase in eukaryotic species (Aslanidis et al. (1989) J. Bacteriol. 171). : 6753-63). The melibiase protein of the present invention includes the protein of SEQ ID NO: 198.

  Carbohydrate utilization related proteins also include, but are not limited to, the following types of enzymes:

  Aldose 1-epimerase (EC: 5.1.3.3) (Mutarotase) (PFAM Accession No. PF01263) is involved in anomeric interconversion of α-form and β-form in D-glucose and other aldoses Is an enzyme. Methods for measuring aldose 1-epimerase activity are known in the art (see, eg, Majumdar et al. (2004) Eur. J. Biochem. 271: 753-9). The aldose 1-epimerase protein of the present invention includes the protein of SEQ ID NO: 200.

  Enolase (2-phospho-D-glycerate hydrolase) is the main glycolytic enzyme that catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate. Methods for measuring phosphopyruvate hydratase activity are known in the art (see, eg, Fox et al. (1995) Plant Physiol. 109: 433-43). SEQ ID NO: 254 contains the enolase C-terminal TIM barrel domain (PFAM accession number PF00113) and the enolase N-terminal domain (PFAM accession number PF03952).

Fructose-bisphosphate aldolase (
1162279228773_2.13
) Is a glycolytic enzyme that catalyzes the reversible cleavage of aldol and the condensation of fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde triphosphate. There are two classes of fructose bisphosphate aldolases with different catalytic mechanisms. Class II aldolase (PFAM accession number PF01116) (Marsh and Lebherz (1992) Trends Biochem. Sci. 17: 110-3) is a dimeric enzyme mainly present in prokaryotes and fungi, Requires a divalent metal ion (usually zinc). This family includes the E. coli galactitol operon protein gatY and the E. coli N-acetylgalactosamine operon which catalyze the conversion of tagatose 1,6-bisphosphate to glycerone phosphate and D-glyceraldehyde triphosphate. The protein agaY is also included. In the first half of these enzyme sequences, there are two histidine residues known to be involved in binding to zinc ions. Methods for measuring fructose bisphosphate aldose activity are known in the art (see, eg, Alefounder et al. (1989) Biochem. J. 257: 529-534). The fructose bisphosphate aldolase class II protein of the present invention includes the protein of SEQ ID NO: 260.

  Galactose-1-phosphate uridyltransferase catalyzes the conversion of UTP and α-D-galactose monophosphate to UDP-glucose and pyrophosphate during galactose metabolism. The C-terminal domain (PFAM accession number PF02744) represents galactose-1-phosphate uridyltransferase. The N-terminal domain (PFAM accession number PF01087) represents the N-terminus of galactose-1-phosphate uridyltransferase. SEQ ID NO: 202 includes both of these domains. Methods for measuring UTP-hexose-1-phosphate uridyltransferase activity are known in the art (see, for example, Lobelle-Rich and Reeves (1983) Mol. Biochem. Parasitol. 7: 173-182).

  Galacto- (EC: 2.7.1.6), homoserine (EC: 2.7.1.39), mevalonate (EC: 2.7.1.36) and phosphomevalonic acid (EC: 2) 7.4.2) each kinase has a conserved region rich in Gly / Ser that is thought to be involved in ATP binding at the N-terminal portion of each. SEQ ID NO: 204 is a member of the virtual ATP binding protein family of GHMP kinase (PFAM Accession No. PF00288). Methods for measuring kinase activity are known in the art (see, eg, Tsay and Robinson (1991) Mol. Cell Biol. 11: 620-31).

  The NAD-dependent epimerase / dehydratase family (PFAM accession number PF01370) protein utilizes NAD as a cofactor. This family of proteins uses nucleotide-sugar substrates in various chemical reactions (Thoden et al. (1997) Biochemistry 36: 6294-304). NAD-dependent epimerase / dehydratase proteins of the present invention include the protein of SEQ ID NO: 216.

  Lantibiotic and non-lantibiotic bacteriocins are synthesized as precursor peptides with an N-terminal extension (leader peptide) that is cleaved during maturation. Most non-lantibiotics and some lantibiotics have a so-called double-glycine type leader peptide. Such leader peptides are common in that they have a common consensus sequence and a processing site containing two glycine-conserved residues at positions 1 and 2. The double glycine type leader peptide is not associated with an N-terminal signal sequence that allows the protein to pass through the cell membrane via the sec pathway. The processing site of the double glycine type leader peptide is also different from the normal signal peptidase cleavage site, suggesting the involvement of another processing enzyme. Peptide bacteriocins cross the cell membrane by a dedicated ATP binding cassette (ABC) transporter. The ABC transporter is a mature protease and its proteolytic domain is present at the N-terminal part (Havarstein et al. (1995) Mol. Microbiol. 16: 229-40). The peptidase C39 family (PFAM accession number PF03412) domain is found in a wide variety of ABC transporters. However, the putative catalytic cysteine and catalytic histidine are not conserved in all members of this family. The peptidase C39 family protein of the present invention includes the protein of SEQ ID NO: 144. Peptidase activity can be assessed by measuring hydrolytic activity (eg, Sasaki et al. (1995) J. Dairy Res. 62: 601-610, and Machuga and Ives (1984) Biochim. Biophys. Acta 789: See 26-36).

  The pfkB family carbohydrate kinase family (PFAM accession number PF00294) includes various carbohydrate kinases and pyrimidine kinases. This family includes phosphomethylpyrimidine kinase (EC: 2.7.4.7), fructokinase (EC: 2.7.1.4), and ribokinase (EC: 2.7.1.15) ( rbsK gene). This enzyme is part of the thiamine pyrophosphate (TPP) synthesis pathway, and TPP is an important cofactor for many enzymes. Methods for measuring kinase activity are known in the art (see, eg, Sato et al. (1993) J. Gen. Microbiol. 139: 921-7). The carbohydrate kinase proteins belonging to the pfkB family of the present invention include the proteins of SEQ ID NOs: 60, 186, 224 and 238.

  Converting the phosphoryl group of ATP using an enzyme as a catalyst is an important reaction in a wide variety of biological processes. One enzyme that utilizes this reaction is phosphofructokinase (PFK) (PFAM Accession No. PF00365), which is an important regulatory step in the glycolytic pathway from fructose 6-phosphate to fructose-1 Catalyze phosphorylation to 6,6-bisphosphate. PFK is about 300 amino acids long, and structural studies of bacterial enzymes have shown that PFK has two similar (α / β) lobes. One such lobe is involved in ATP binding and the other contains both a substrate binding site and an allosteric site (a regulatory binding site different from the activation site but affecting the enzyme activity). Have. The same trimeric subunit takes two different structures, and the products from such structures are very different. One such structure is in a “closed” state, where the bound magnesium ion crosslinks to the phosphoryl group of the enzyme product (ADP and fructose-1,6-bisphosphate) and the other structure is “open ( open) "state, and magnesium ions crosslink only to ADP. Such a structure is thought to be a successive step in the reaction pathway that requires a subunit closure to bring the two molecules into a reactive proximity (Shirakihara and Evans (1988) J. Mol. Biol. 204 : 973-94). Methods for measuring the activity of 6-phosphofructokinase are known in the art (see, eg, Wegener and Krause (2002) Biochem. Soc. Trans. 30: 264-70). The phosphofructokinase protein of the present invention includes the protein of column number 256.

  Phosphoglucose isomerase (EC: 5.3.1.9) (PGI) (PFAM accession number PF00342) is a dimeric enzyme that catalyzes the reversible isomerization of glucose 6-phosphate and fructose 6-phosphate. is there. PGI is involved in different pathways, is involved in glycolysis in most higher organisms, is involved in gluconeogenesis in mammals, is involved in carbohydrate biosynthesis in plants, and in some bacteria fructose is entoner Provides a route to enter the Entner-Doudouroff route. PGI, a multifunctional protein, is also a neuroleukin (a neurotrophic factor that promotes neuronal differentiation), an autocrine motility factor (a cytokine that is secreted by a tumor and regulates cell motility), a differentiation mediator and a mature mediator It is also known as a serine proteinase inhibitor that binds to myofibrils and plays different roles inside and outside the cell. PGI catalyzes the second step of glycolysis intracellularly and functions extracellularly as a nerve growth factor and cytokine. Methods for measuring glucose 6-phosphate isomerase activity are known in the art (see, eg, Nozue et al. (1996) DNA Seq. 6: 127-35). The phosphoglucose isomerase protein of the present invention includes the protein of SEQ ID NO: 252.

  Phosphoglycerate kinase (EC: 2.7.2.3) (PGK) (PFAM accession number PF00162) is an enzyme that catalyzes the formation of ADP from ATP and the formation of ATP from ADP. In the second step in the second stage of glycolysis, 1,3-diphosphoglyceric acid is converted to 3-phosphoglyceric acid to form one ATP molecule. If the reverse occurs, one ADP molecule is formed. This reaction is an important reaction for most cells due to the production of ATP in aerobic organisms, fermentation in anaerobic organisms, and carbon solidification in plants. All organisms have PGK, and their sequences have been highly conserved during evolution. PGK has two domains corresponding to the N-terminal and C-terminal of the protein, and exists as a monomer whose domains are almost the same size (the last 15 residues on the C-terminal side are folded back to the N-terminal). Return to domain). 3-phosphoglycerate (3-PG) binds to the N-terminus, and the nucleotide substrates MgATP or MgADP bind to the C-terminus of 3-PG. Such two-domain extended structures result in a large “hinge-bending” structural change as found in hexokinase (Kumar et al. (1999) Cell Biochem. Biophys. 31 : 141-64). At the core of each domain is a β sheet that is surrounded by an α helix and is a parallel 6 strand. Domain 1 has six strands of parallel β-sheets arranged in the order of 342156, and domain 2 has six strands of parallel β-sheets arranged in the order of 321456. Analysis of reversible unfolding of the yeast phosphoglycerate kinase showed that the two lobes were folded independently, indicating that one of the domains was folded. Consistent with the presence of intermediates in the folding pathway (Yon et al. (1990) Biochimie 72: 417-29). Methods for measuring phosphoglycerate kinase activity are known in the art (see, eg, Pal et al. (2004) Biochim. Biophys. Acta. 1699: 277-80). The phosphoglycerate kinase protein of the present invention includes the protein of SEQ ID NO: 250.

  Phosphoglycerate mutase (EC: 5.4.2.1) (PGAM) and bisphosphoglycerate mutase (EC: 5.4.2.4) (BPGM) are structurally related enzymes. Yes, it catalyzes a reaction involving the conversion of a phosphate group between three carbon atoms of phosphoglyceric acid. All of the above enzymes are isomerized from 2-phosphoglycerate (2-PGA) to 3-phosphoglycerate (3-PGA) using 2,3-diphosphoglycerate as a reaction primer, and 3-PGA. Can catalyze three different reactions with different specificities: synthesis from 2,3-DPG to 1,3-DPG, and degradation from 2,3-DPG to 3-PGA. Yes (phosphatase EC: 3.1.3.13 activity). Numerous other proteins also belong to this family, for example, bifunctional enzymes that catalyze the synthesis and degradation of fructose-2,6-bisphosphate and bacterial α-ribazole-5′-phosphate phosphatase, including cobalamin ( 6-phosphofructo-2-kinase / fructose-2,6-bisphosphatase involved in the biosynthesis of cobalamin) also belongs to this family. Methods for measuring the catalytic activity of the enzymes are known in the art (see, eg, Rigden et al. (2001) Protein Sci. 10: 1835-46). The phosphoglycerate mutase family (PFAM accession number PF00300) protein of the present invention includes the protein of SEQ ID NO: 244.

  Pyruvate kinase (EC: 2.7.1.40) (PK) catalyzes the conversion of phosphoenolpyruvate to pyruvate with phosphorylation of ADP to ATP, the final step in glycolysis. Most bacteria and lower eukaryotes have one form of this enzyme, with the exception of certain bacteria (eg, E. coli), which have two isozymes. All isozymes are thought to be tetramers consisting of the same subunit of about 500 residues. PK, together with phosphofructokinase and hexokinase, helps control the rate of glycolysis. Methods for measuring pyruvate kinase activity are known in the art (see, for example, Boles et al. (1997) J. Bacteriol. 179: 2987-93). The pyruvate kinase α / β domain (PFAM accession number PF02887) protein in the present invention includes the protein of SEQ ID NO: 258. The pyruvate kinase barrel domain (PFAM accession number PF00224) in the present invention includes the protein of SEQ ID NO: 258.

  A number of enzymes require thiamine pyrophosphate (TPP) (vitamin B1) as a cofactor. Some of these enzymes have been shown to be structurally related (Green (1989) FEBS Lett. 246: 1-5). The thiamine pyrophosphate enzyme central domain (PFAM accession number PF00205) contains two Rossman folds. SEQ ID NO: 232 includes not only the N-terminal TPP binding domain of thiamine pyrophosphate enzyme (PFAM accession number PF02776), but also the central domain of thiamine pyrophosphate enzyme.

  The enzyme 3β-hydroxysteroid dehydrogenase / 5-ene-4-ene isomerase (3β-HSD) is responsible for the oxidation and isomerization of the 5-ene-3β-hydroxypregnene and 5-ene-hydroxyandrostene steroid precursors. Catalyze to the corresponding 4-ene-keto steroids necessary for the formation of all steroid hormone classes. The 3-β-hydroxysteroid dehydrogenase / isomerase family (PFAM accession number PF01073) protein of the present invention includes the protein of SEQ ID NO: 216. Methods for measuring 3-β-hydroxy-delta5-steroid dehydrogenase activity are known in the art (see, eg, Moisan et al. (1999) J Clin Endocrinol Metab. 84: 4410-25).

  Dihydroxyacetone kinase (glycerone kinase) (EC: 2.7.1.29) catalyzes the phosphorylation of glycerone in the presence of ATP, resulting in glycerone phosphate in the glycerol utilization pathway. The Dak1 domain (PFAM accession number PF02733) is a kinase domain of the dihydroxyacetone kinase family. The Dak1 domain protein of the present invention includes the protein of SEQ ID NO: 190. The DAK2 domain (PFAM accession number PF02734) is a virtual phosphatase domain in the dihydroxyacetone kinase family. The Dak2 domain protein of the present invention includes the protein of SEQ ID NO: 192. Methods for measuring glycerone kinase activity are known in the art (see, eg, Sellinger and Miller (1957) Fed. Proc. 16: 245-246).

  The biosynthesis of disaccharides, oligosaccharides and polysaccharides is associated with the action of hundreds of different glycosyltransferases. Glycosyltransferases are enzymes that catalyze the transfer of a sugar component from an activated donor molecule to a specific acceptor molecule to form a glycosidic bond. Family classification based on the sequence differences of glycosyltransferases using nucleotide diphospho sugars, nucleotide monophospho sugars, sugar phosphates (EC: 2.4.1), and related proteins is shown. The same 3D folding is expected to occur in each family. Because 3D structures are better conserved than sequences, some families defined on the basis of sequence similarity have similar 3D structures, so that "clan" It is formed. Members of the group 1 family of glycosyltransferases (PFAM accession number PF00534) transfer UDP, ADP, GDP or CMP-linked sugars. Bacterial enzymes are involved in a variety of biosynthetic processes, including exopolysaccharide biosynthesis, lipopolysaccharide core biosynthesis, and mucus polysaccharide colanic acid biosynthesis. The group 1 protein of the glycosyltransferase of the present invention includes the protein of SEQ ID NO: 328.

  The kinase proteins of the present invention include the proteins of SEQ ID NOs: 224, 230, 190, 192, 186, and 238. The hydrolyzed proteins of the present invention include SEQ ID NOs: 10, 46, 50, 60, 100, 108, 196, 198, 200, 202, 204, 210, 212, 214, 216, 218, 220, 222, 226, 276. , 292, 342, 344, 346, 356, 358, 362, and 364 proteins. The proteins of the invention involved in metabolism include the proteins of SEQ ID NOs: 60, 186, 200, 202, 216, 218, 228, 342, 344, 346, 356, and 358. The glycolysis proteins of the present invention include the proteins of SEQ ID NOs: 242, 244, 246, 248, 250, 252, 254, 256, 258, 260. The glycogen metabolizing proteins of the present invention include the proteins of SEQ ID NOs: 240, 324, 326, 328, 330, and 332 The proteins of the invention involved in EPS metabolism include the proteins of SEQ ID NOs: 348, 350, 352, and 354.

  Methods for cloning and expressing carbohydrate utilization-related proteins in microorganisms and plants, and methods for assessing the function of such proteins are known in the art (eg, de Vos (1996) Antonie van Leeuwenhoek 70: 223-242 Yeo et al. (2000) Mol. Cells 10: 263-268; Goddijn et al. (1997) Plant Physiol. 113: 181-190). For example, enzyme functions, fermentation assays, and transport assays assess protein function associated with primary and secondary transport systems. The function of the group transfer system-related protein is evaluated by, for example, a sugar phosphorylation assay. For example, Russell et al. (Russell et al. (1992) J. Biol. Chem. 267: 4631-) was identified and expressed in Escherichia coli, the gene of the primary transport system (msm) of Streptococcus mutans. 4637) and two types of Lactobacillus bulgaricus secondary transport system (lactose) cloned and expressed in Escherichia coli, Leong-Morgenthaler et al. (Leong-Morgenthaler et al. (1991 ) J. Bacteriol. 173: 1951-1957) and Vaughan et al. (Vaughan et al. (Vaughan et al. (Vaughan et al. (Vaughan et al. 1996) Appl. Env. Microbiol. 62: 1574-1582) and two PTS genes in Lactococcus lactis were identified, cloned and expressed in E. coli and Lactobacillus lactis. et al. (de Vos et al. (1990) J. Biol. Chem. 265: 22554-22560) and the ScrA gene of Streptococcus mutans were cloned into Escherichia coli. (Sato et al. (1989) J. Bacteriol. 171: 263-271) and the gene encoding the lactose-specific enzyme II of Lactobacillus casei were cloned into E. coli and expressed. Chassy (Alpert and Chassy (1990) J. Biol. Chem. 265: 22561-22568) and Boyd et al., A gene encoding HPr and enzyme I of the PTS transport system of Streptococcus mutans cloned into E. coli and expressed. (Boyd et al. (1994) Infect. Immun. 62: 1156-1165) and overexpression of the trehalose biosynthesis genes otsA and otsB in E. coli, the tolerance of transgenic plants to abiotic stress And improved productivity, Garg et al. (Garg et al. (2002) Proc. Natl. Acad. Sci. USA 99: 15898-15903) and multi-drug resistant proteins are expressed as drug pumps See Grinius and Goldberg (Grinius and Goldberg (1994) J. Biol. Chem. 269: 29998-30004) which has been shown to function.

  By expressing one or more carbohydrate utilization-related proteins, it becomes possible to modify the organism's ability to accumulate carbohydrates in the cytoplasm of cells. For example, by introducing or overexpressing an enzyme involved in carbohydrate catabolism without expressing the relevant transport protein, such a carbohydrate could be accumulated in the cytoplasm. Alternatively, introduction or overexpression of a carbohydrate transport-related protein will enhance carbohydrate transport to the external environment. Methods for introducing or expressing a carbohydrate utilization-related gene into an organism are known in the art. The accumulation of carbohydrates in the cells can be assessed, for example, by chromatographic methods or enzymatic assays. See, for example, Chaillou et al. (1998) J. Bacteriol. 180: 4011-4014 and Goddijn et al. (1997), supra.

  By expressing one or a plurality of carbohydrate utilization-related proteins, it becomes possible to modify the organism's ability to use carbohydrates as energy sources or to generate them as energy sources. Methods for cloning and expressing carbohydrate utilization-related proteins in organisms and methods for evaluating the function of such proteins are known in the art (eg de Vos (1996) Antonie van Leeuwenhoek 70: 223-242; Hugenholzet al. (2002) Antonie van Leeuwenhoek 82: 217-235). For example, in order to enhance the utilization of lactose, a gene for lactose metabolism is introduced into bacteria to produce a product more suitable for people who are not resistant to lactose (the above-mentioned Hugenholz et al. (2002)). Further, the bacterium so modified may be further modified. For example, natural metabolism is provided by inhibiting glucose metabolism so that glucose is not degraded but released from the cell to the medium. For example, see Hugenholz et al. (2002) above. Alternatively, as well as the α-phosphoglucomutase gene, introducing a gene for galactose metabolism to increase the ability of microorganisms to ferment galactose can help prevent high levels of galactose consumption (Hugenholz et al. 2002) supra; Hirasuka and Li (1992) J. Stud. Alcohol 62: 397-402). One gene associated with galactose metabolism is α-galactosidase. Since monogastric animals cannot degrade raffinose-type sugars, the expression of α-galactosidase is useful for removing raffinose-type sugars from fermentation products (Hugenholz et al. (2002) above). When a bacterial gene of mannitol-1-phosphate dehydrogenase (mtlD) was expressed in tobacco plants, mannitol was successfully synthesized and accumulated (Tarczynski et al. (1992) Proc. Natl. Acad. Sci. USA 89: 2600-2604 ).

  The function of various carbohydrate utilization-related proteins is assessed, for example, by microbial growth assays, transport assays, enzyme assays, or analysis by chromatographic methods and NMR. See, for example, Djordjevic et al. (2001) J. Bacteriol. 183: 3224-3236; Chaillou et al. (1998) J. Bacteriol. 180: 4011-4014; and Tarczynski et al. (1992) above.

  In general, permeases, membrane-bound enzymes, and regulatory factors such as transcriptional repressors or antiterminators need to be expressed in cells in order to make optimal use of carbohydrates. Transcriptional antiterminator function can be measured by antitermination activity in the reporter system (see, eg, Alpert and Siebers (1997) J. Bacteriol. 179: 1555-1562). The function of repressors such as lacR can be measured by enzyme activity or proliferation assays (eg, van Rooijen et al. (1993) Protein Eng. 6: 201-206; van Rooijen and de Vos (1990) J. Biol. Chem). 265: 18499-18503).

  Bacterial regulatory proteins of the present invention include proteins in SEQ ID NOs: 8, 38, 98, 104, 118, 150, 178, 180, 182, 184, 188, 266, 290, 304, and 336.

  Bacterial regulatory protein lacI family (PFAM accession number PF00356) proteins in the present invention include the proteins of SEQ ID NOs: 38, 98 and 182. The bacterial regulatory protein gntR family (PFAM accession number PF00392) in the present invention includes the protein of SEQ ID NO: 106. Bacterial regulatory helix-turn-helix protein AraC family (PFAM Accession No. PF00165) proteins in the present invention include the protein of SEQ ID NO: 118. Bacterial regulatory protein deoR family (PFAM accession number PF00455) proteins include the proteins of SEQ ID NOs: 188 and 336.

  The PRD domain (PTS regulatory domain) (PFAM accession number PF00874) is a phosphorylable regulatory domain found not only in activators such as MtlR and LevR, but also in bacterial transcription antiterminators belonging to the BglG family. The PRD domain is phosphorylated at a conserved histidine residue. Proteins with PRD are involved in the regulation of the catabolic operon of Gram-positive and Gram-negative bacteria, characterized by RNA (activator) RNA (anti-terminator (CAT_RBD) CAT-RBD) Often it is made by a replicating PRD molecule that is phosphorylated by a sugar phosphotransferase system (PTS) corresponding to the availability of a carbon source at a conserved histidine residue or at a conserved histidine residue. Such phosphorylation modifies the stability of the dimeric protein, thereby changing the binding activity of the effector domain to RNA or DNA. This is a family of bacterial proteins related to the E. coli bglG protein. The E. coli bglG protein promotes positive regulation of the β-glucoside (bgl) operon by functioning as a transcriptional antiterminator (Houman et al. (1990) Cell 62: 1153-63). bglG is an RNA binding protein that recognizes a specific sequence located immediately upstream of two termination sites within the operon. The activity of bglG is suppressed by phosphorylation by bglF (EII-bgl), a β-glucoside PTS permease (Amster-Choder and Wright (1990) Science 249: 540-2). bglG is very similar to other proteins thought to act as transcription antiterminators. The PRD domain-containing protein of the present invention includes the proteins of SEQ ID NOs: 8 and 266.

  The AraC-like ligand binding domain family (PFAM accession number PF02311) occupies the arabinose binding domain and dimerization domain in the bacterial gene regulatory protein AraC. This domain is found with the helix-turn-helix (HTH) DNA binding motif HTARAraC. The relationship between such a domain and the cupin domain is low. The AraC-like ligand binding domain family proteins in the present invention include the protein of SEQ ID NO: 118.

    The CAT RNA binding domain (PFAM accession number PF03123) is found at the amino terminus of the transcription antiterminator protein family. This domain is called CAT (co-antiterminator) and forms a dimer in a known structure. Transcriptional antiterminators of the BglG / SacY family are regulatory proteins that promote the induction of glucose metabolism operons in Gram positive and Gram negative bacteria. Such proteins, when activated, bind to specific targets in nascent mRNA, thus preventing failure of dissociation of RNA polymerase from the DNA template (Declerck et al. (1999) J. Mol. Biol. 294: 389-402 ). CAT RNA binding domain proteins of the present invention include the proteins of SEQ ID NOs: 8 and 266.

  SEQ ID NO: 184 is a member of the HPr serine kinase N-terminal family (PFAM accession number PF02603) and is also a member of the HPr serine kinase C-terminal family (PFAM accession number PF07475). The N-terminal family represents the N-terminal region of Hpr serine / threonine kinase PtsK. The C-terminal family represents the C-terminal kinase domain of Hpr serine / threonine kinase PtsK. This kinase is a sensor in the multicomponent phosphate relay system that controls the inhibition of carbon catabolism in bacteria (Marquez et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99: 3458-63). This kinase is unique in that it recognizes the tertiary structure of its target and is a member of a novel family that is not associated with any of the protein kinases reported so far. As a result of X-ray analysis of the full-length crystal enzyme of Staphylococcus xylosus at a resolution of 1.95A, this enzyme consists of two clearly separated domains, which have three propellers. Is associated with a hexameric structure similar to These wings are each formed with two N-terminal domains, and a compact central hub is associated with a C-terminal kinase domain (Reizer et al. (1998) Mol. Microbiol. 27: 1157-69). ).

  The sequences of the present invention also modify the ability of the organism to change the flavor and texture of food. Modifying glucose metabolism to produce alternative sugars is one approach to changing flavor and texture characteristics. If the lactate dehydrogenase gene is disrupted by simultaneously expressing the mannitol operon or sorbitol operon gene, mannitol and sorbitol are produced (Hugenholz et al. (2002)). Production of diacetyl in the fermentation process results in butter aroma, which is enhanced by destroying lactate dehydrogenase or by overexpressing NADH oxidase while destroying α-aceto lactate decarboxylase (Hugenholz and Kleerebezem, (1999) supra; Hugenholtz et al. (2000) Appl. Environ. Microbiol. 66: 4112-4114). Alternatively, the production of diacetyl was also increased by overexpression of α-acetolactate synthase or acetohydroxy acid synthase with the destruction of α-acetolactate decarboxylase (Swindell et al. (1996) Appl. Environ. Microbiol 62: 2641-2643; Platteeuw et al. (1995) Appl. Environ. Microbiol. 61: 3967-3971). Overexpression of alanine dehydrogenase produces alanine instead of lactic acid, and provides a fermented food with a taste-enhancer and sweetener (Hols et al. (1999) Nat. Biotechnol. 17: 588 -592).

  Also encompassed by the invention are methods of altering the ability of an organism to produce modified carbohydrates. Such methods include introducing at least one of the nucleotide sequences of the present invention into an organism. Also encompassed by the present invention are methods for providing modified carbohydrates, such methods comprising contacting the modified carbohydrate with a polypeptide of the present invention. Methods for producing modified carbohydrates are known in the art. See, for example, Kim et al. (2001) Biotechnol. Prog. 17: 208-210.

  The sequences of the present invention also modify an organism's ability to survive in the food system or mammalian gastrointestinal tract, or alter the stability and viability of an organism under food processing and storage. For example, increasing the production of trehalose maintains the freshness and flavor of the fermented product (see, eg, www.nutracells.com). Trehalose may also be useful for preventing diseases caused by protein aggregation and the pathological structure of proteins such as Creutzfeldt-Jakob disease. In plants, trehalose accumulation helps protect against environmental stresses such as drought, salt, cold (eg, Jang et al. (2003) Plant Physiol. 131: 516-524; Penna (2003) Trends Plant Sci. 8: 355-357; Garg et al. (2002) Proc. Natl. Acad. Science 99: 15898-15903; see Yeo et al. (2000) above). Furthermore, transformation of plants with the fructosyltransferase gene was able to increase the accumulation of fractans in plants (van der Meer et al. (1994) Plant Cell 6: 561-570). In addition to the role of stored carbohydrates, fructans also provide resistance to dry and cold conditions (Pontis and del Campillo (1985) “Fructans” in Biochemistry of Storage Carbohydrates in Green Plants, Day and Dixon, eds (London: Academic Press), pp. 810-816; Pilon-Smits et al. (1995) Plant Physiol. 107: 125-130). The bacterial gene mannitol-1-phosphate dehydrogenase is also expressed in plants, thereby producing mannitol, which is believed to confer beneficial properties on osmotic regulation and neutralization of hydroxy radicals. (Tarczynski et al. (1992) above).

  The multidrug transporter sequences of the present invention allow an organism to survive even when contacted with an antimicrobial polypeptide or other toxin. This is considered to be because the ability to excrete the drug or toxin out of the cell is enhanced.

Variants of the nucleotide sequence of the present invention that retain or modify the ability to transport carbohydrates or toxins into and out of cells, as well as variants that retain or modify the ability to accumulate or utilize carbohydrates are also encompassed by the present invention. Methods for preparing and testing carbohydrate utilization-related proteins or multidrug transporter protein variants are known in the art. For example, variants of secondary transport system proteins (melibiose and lactose) with altered substrate specificity were isolated, constructed and tested Poolman et al. (Poolman et al. (1996) Mol. Microbiol. 19: 911-911 ) checking. In these mutants, sugar transport is not conjugated to cation symport. For example, the above-mentioned Djorovevic et al. (2001), and the cold cooling of the β-galactosidase gene of Lactobacillus delbrueckii subsp. See also Adams et al. (Adams et al. (1994) J. Biol. Chem. 269: 5666-5672), who created and characterized a sensitive variant. These mutant genes are useful for preventing oxidation of fermented products under cold storage due to a decrease in V _max at low temperatures (Mainzer et al. (1990) “Pathway engineering of Lactobacillus bulgaricus for improved yoghurt, In Yoghurt; Nutritional and Health Properties, Chandan, ed., (National Yoghurt Association, Virginia, US), pp. 41-55). See also Bettenbrock et al. (Bettenbrock et al. (1999) J. Bacteriol. 181: 225-230), which isolated a mutant with a modified galactose-specific PTS gene. See also van Rooijen et al. (1990), above, who isolated a variant of the lacR repressor that does not affect activity. Furthermore, Kroetz et al. (Kroetz et al. (2003) Pharmacogenetics 13: 481-94) which analyzed the polymorphism of the human MDR1 gene, and Mitomo et al. (Mitomo et al.) Which analyzed the ABC transporter ABCG2 variants. (2003) Biochem. J. 373: 767-74).

  Any of the above modifications may be combined with other metabolic modifications engineered with or suggested for lactic acid bacteria. Such metabolic alterations include vitamin B production such as folic acid (B11), riboflavin (B2), cobalamin (B12), polyol production, sucrose as a sweetener, low calorie sugar as an alternative to lactose, glucose or fructose, Production of tagatose as a sucrose substitute, production of various exopolysaccharides, inhibition of glucose metabolism to provide a natural sweetening effect, reduction of galactose production, production of foods with low α-galactoside content such as stachyose and raffinose Increased production of trehalose with improved food preservation and potential for disease prevention (Hugenholz et al. (2002); van Roojen et al. (1991) J. Biol. Chem. 266 : 7176-7178).

  Also provided are methods for removing and modifying undesirable carbohydrates in food or chemical products. This method involves contacting the product with a purified polypeptide of the invention. Assay methods for carbohydrate removal or modification are known in the art.

  The following examples are for illustrative purposes and are not intended to be limiting.

Example 1 [Results of BlastP in Consideration of Gap for Each Amino Acid Sequence]
Sequence alignment with BlastP considering the gap showed the following. That is, SEQ ID NO: 2 (144 amino acids) is homologous to PTS-based mannose-specific factor IIAB (Accession Number: NP — 4694888.1; NC — 003212) obtained from Listeria innocua. PTS-based mannose-specific factor IIAB (Accession Number: NP_463629.1; NC — 003210) with approximately 61% identity to the protein, whose 1-140 amino acids were obtained from Listeria monocytogenes Mannose-specific phosphotransferase system component IIAB (accession number: NP_1), which has about 60% identity with a homologous protein and whose 1 to 139 amino acids are obtained from Clostridium acetobutylicum 49230.1; NC — 001988), a PTS protein (Accession Number: NP — 561737.1; NC — 00366) whose 1 to 139 amino acids were obtained from Clostridium perfringens. ) And about 2-141 amino acids of which mannose-specific phosphotransferase system component IIAB (accession number: NP — 269761.1; NC — 002737) obtained from Streptococcus pyogenes It was shown to have about 50% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 4 (123 amino acids) is a phosphotransferase system (PTS) lichenan-specific enzyme IIA component (accession number NP_4711165.1; NC_003212) whose 20-109 amino acids were obtained from Listeria innocure. A protein having about 60% identity with a homologous protein and 20 to 110 amino acids of which is homologous to the cellobiose phosphotransferase enzyme IIA component (Accession No: NP — 472161.1; NC — 003212) obtained from Listeria innocure A cellobiose-specific PTS-based IIA component tongue from which 1-101 amino acids were obtained from Lactococcus lactis subsp. Lactis It has about 46% identity with EC (EC 2.7.1.69) (accession number: NP — 266570.1; NC — 002662), and its 9 to 112 amino acids are obtained from Bacillus halodurans. Furthermore, PTS cellobiose-specific enzyme IIA component protein (accession number: NP — 241776.1; NC — 002570) has about 44% identity, and its 16 to 112 amino acids were obtained from Streptococcus pyogenes, It was shown to have about 51% identity with a protein homologous to enzyme III (accession number: NP_607437.1; NC_003485).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 6 (161 amino acids) is a β-glucoside-specific transport protein (BglS) (accession number: gb | AAD228228.1) whose 6-143 amino acids were obtained from Enterococcus faecium; AF121254) with about 53% identity, 13 to 159 amino acids obtained from Streptococcus pneumoniae, PTS IIABC component protein (accession number: NP_345256.1; NC_003028) and about 48 PTS glucose-specific enzyme IIABC component protein (accession number: NP — 358262.1; NC — 00) with 13% identity and 13 to 159 amino acids obtained from Streptococcus pneumoniae 098) and about 13% of its amino acids are about 13 to 159 amino acids from a protein homologous to the PTS enzyme IIA component protein (accession number: NP — 608025.1; NC — 003485) obtained from Streptococcus pyogenes 46% identity, and 13 to 159 amino acids are approximately 46% of a protein homologous to the PTS enzyme IIA component (Accession Number: NP — 269950.1; NC — 002737) obtained from Streptococcus pyogenes It was shown to have identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 8 (291 amino acids) is approximately 36% of the transcription antiterminator (licT) (accession number: p | P39805 | LICT_BASU) whose 11-282 amino acids were obtained from Bacillus subtilis. The amino acids 11 to 282 have about 36% identity with the transcriptional anti-terminator (BglG family) (accession number: NP_391787.1; NC_000964) obtained from Bacillus subtilis, The 11-282 amino acids have about 37% identity with the protein involved in the positive regulation of the bgl operon (accession number: NP_418179.1; NC_000913) obtained from E. coli, and the 11-282 amino acids are , Erwinia Krisan It has about 33% identity with β-glucoside operon antiterminator (accession number: p | P26211 | ARBG_ERWCH) obtained from Erwinia chrysanthemi, and its 9-288 amino acids are from Clostridium acetobutylicum The obtained transcription antiterminator (licT) (accession number: NP — 347062.1; NC — 003030) was shown to have about 34% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 10 (480 amino acids) is homologous to phospho-β-glucosidase phospho-β-glucosidase (accession number: NP — 43849.1; NC — 003210) obtained from Listeria monocytogenes. About 58% identity with a protein homologous to phospho-β-glucosidase (Accession No: NP — 46969.1; NC — 003212) obtained from Listeria Inocure. Of which 7-473 amino acids have about 57% identity with 6-phospho-β-glucosidase (NP_3477379.1; NC_003030) obtained from Clostridium acetobutylicum, 473 amino acids It has about 57% homology with 6-phospho-β-glucosidase (accession number: sp | Q46130 | ABGA_CLOLO) obtained from Clostridium longisporum, and 1 to 473 amino acids thereof are Bacillus. -It was shown to have about 55% identity with β-glucosidase (accession number: NP_391805.1; NC_000964) obtained from subtilis.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 12 (625 amino acids) is approximately 38% identical to β-glucoside permease II ABC component protein (accession number: NP — 268836.1; NC — 002737) whose 1 to 624 amino acids were obtained from Streptococcus pyogenes. 1 to 624 amino acids have about 38% identity with β-glucoside permease IIABC component protein (Accession Number: NP — 606826.1; NC — 003485) obtained from Streptococcus pyogenes, 1-605 amino acids obtained from Streptococcus pneumoniae, a phosphotransferase-based sugar-specific EII component protein (accession number: NP — 35809.9; NC — 00309) ), About 1 to 605 amino acids of which were obtained from Streptococcus pneumoniae with a PTS β-glucoside-specific IIABC component protein (accession number: NP — 345091.1; NC — 003028) and about 38 % Of its identity and about 1 to 622 amino acids with the PTS β-glucoside specific enzyme IIABC component protein (accession number: NP — 241162.1; NC — 002570) obtained from Bacillus halodurans. % Identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 14 (675 amino acids) is approximately 50% of the PTS β-glucoside-specific IIABC component protein (accession number: NP — 348035.1; NC — 003030) whose 17 to 648 amino acids are obtained from Clostridium acetobutylicum. The 17-656 amino acids have about 50% identity with the PTS β-glucoside specific enzyme IIABC (accession number: NP — 241461.1; NC — 002570) obtained from Bacillus halodurans. About 17% of the amino acid having a homology with the PTS β-glucoside specific enzyme IIABC (accession number: NP — 463560.1; NC — 003210) obtained from Listeria monocytogenes. And its 17-654 amino acids have about 48% identity with PTS-dependent enzyme II (accession number: gb | AAC05713.1; L49336) obtained from Clostridium longisporum, and 13 ~ 654 amino acids have been shown to have about 48% identity with β-glucoside specific EII permease (accession number: gb | AAF8975.1; AF206272) obtained from Streptococcus mutans. It was.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 16 (445 amino acids) has a phosphotransferase system (PTS) protein lichenan-specific enzyme IIC component protein (accession number: NP — 391737.) whose 10 to 443 amino acids are obtained from Bacillus subtilis. 1; NC_000964) with about 41% identity, and its 14 to 442 amino acids are homologous to the PTS IIBC component (ywbA) (accession number: sp | P39584 | YWBA_BASU) obtained from Bacillus subtilis The cellobiose phosphotransferase enzyme IIC component protein (A), which has about 42% identity with the protein and whose 14-441 amino acids are obtained from Bacillus stearothermophilus Session number: sp | Q45400 | PTCC_BACST), a phosphotransferase-based sugar-specific EII component protein (accession number: NP — 358015) having about 41% identity and 12 to 441 amino acids obtained from Streptococcus pneumoniae .1; NC_003098) and having about 41% identity, and 12 to 441 amino acids thereof were obtained from Streptococcus pneumoniae, PTS cellobiose-specific IIC component protein (Accession number: NP_3449993.1; NC_003028). ) And about 40% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 18 (422 amino acids) is approximately 34% of a protein whose 9-417 amino acids are homologous to a phosphotransferase enzyme II (Accession Number: NP_391718.1; NC_000964) obtained from Bacillus subtilis. About 33% identical to the phosphotransferase system (PTS) lichenan-specific enzyme IIC component protein (accession number: NP_391737.1; NC_000964), which has 17-414 amino acids and is obtained from Bacillus subtilis The cellobiose phosphotransferase enzyme IIC component protein (accession number: sp | Q45400 | PTCC_BA) whose 10-417 amino acids were obtained from Bacillus stearothermophilus ST) and a protein having 9-414 amino acids homologous to the PTS cellobiose-specific IIC component (Accession Number: NP — 470241.1; NC — 003212) obtained from Listeria Inocure. PTS cellobiose-specific IIC component protein (celB) (accession number: NP — 0469990.) having about 33% identity and whose 11-415 amino acids were obtained from Borrelia burgdorferi. 1; NC_001903) with about 31% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 20 (130 amino acids) is approximately 33% of the phosphotransferase IIA component protein (accession number: NP — 540949.1; NC — 003317) whose 3 to 124 amino acids are obtained from Brucella melitensis. The 2-102 amino acids have 32% identity with EIIA-mannose protein (Accession No .: gb | AAB04153.1; U28163) obtained from Lactobacillus curvatus. 3 to 96 amino acids have about 32% identity with a protein homologous to a PTS protein (accession number: NP — 56355.1; NC — 00366) obtained from Clostridium perfringens, 123 An amino acid has about 25% identity with a protein homologous to a PTS protein (Accession No .: NP_561737.1; NC_003366) obtained from Clostridium perfringens, and 3 to 123 amino acids thereof are It was shown to have about 25% identity with the mannose-specific phosphotransferase system component IIAB (accession number: NP — 149230.1; NC — 001988) obtained from Clostridium acetobutylicum.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 22 (162 amino acids) has a PTS enzyme IIBC component protein (galactitol / fructose-specific) (Accession number: NP — 349560.1; NC — 003030), 8 to 159 amino acids obtained from Clostridium acetobutylicum. ), About 7 to 158 amino acids, obtained from Streptococcus pneumoniae, a phosphotransferase-based sugar-specific EII component protein (accession number: NP_358156.1; NC_003098) and about 36 % PTS-based IIA component (accession number: NP_345152.1; NC_003028) whose 7-158 amino acids were obtained from Streptococcus pneumoniae A GatA protein (accession number: gb | AAG0997.1; AF248038), which has about 36% identity with a protein homologous to) and whose 20-134 amino acids were obtained from Streptococcus agalactiae It has about 38% identity, and its 16 to 159 amino acids are about the same as the PTS-based galactitol-specific enzyme IIA component protein (accession number: NP — 241058.1; NC — 002570) obtained from Bacillus halodurans. It was shown to have 33% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 24 (466 amino acids) has about 47% identity with PTS cellobiose-specific component IIC (accession number: NP — 347026.1; NC — 003030) whose 30 to 461 amino acids are obtained from Clostridium acetobutylicum. A cellobiose-specific PTS IIC component protein (EC 2.7.6.9) (accession number: NP — 269674.1; NC — 002662) obtained from Lactococcus lactis subsp. Lactis Cellobiose-specific PTS-based IIC component protein (EC 2.7. 1.69) with approximately 45% identity, 82-465 amino acids obtained from Lactococcus lactis subsp. No .: NP — 266572.1; NC — 002662), PTS-based enzyme IIC component (accession number: NP — 2699944.1; NC — 002737), having about 46% identity and 34 to 466 amino acids obtained from Streptococcus pyogenes About 34% of its homologous protein, and its 34-466 amino acids are homologous to the PTS enzyme IIC component (accession number: NP — 608069.1; NC — 003485) obtained from Streptococcus pyogenes It was shown to have about 40% identity with the protein.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 26 (428 amino acids) is approximately 28% of a protein whose 25 to 420 amino acids are homologous to PTS cellobiose-specific enzyme IIC (registration number: NP_472233.1; NC_003212) obtained from Listeria innocure. With 115-415 amino acids having approximately 27% identity with LacE protein (accession number: embCAB025556.1; Z80834) obtained from Lactobacillus casei, 137-425 amino acids Has about 26% identity with a protein homologous to PTS cellobiose-specific enzyme IIC (Accession No .: NP — 472184.1; NC — 003212) obtained from Listeria innocure, and its 137 to 425 amino acids are Listeria・ Monosite Gene The protein has about 26% identity with a protein homologous to the PTS cellobiose-specific enzyme IIC (accession number: NP — 466230.1; NC — 003210) obtained from S. cerevisiae, whose 115 to 415 amino acids are from Lactobacillus casei It was shown to have about 26% identity with the obtained phosphotransferase enzyme II (EC 2.7.1.69) (accession number: pi || B23697).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 28 (475 amino acids) is a cellobiose-specific PTS IIC component protein (EC 2.7.1.69) whose accession number is 10 to 471 amino acids obtained from Lactococcus lactis subspecies lactis (accession number). : NP — 266794.1; NC — 002662), which is about 57% identical, and whose 71-475 amino acids were obtained from Lactococcus lactis subsp. Lactis (EC 2.7.1). .69) (accession number: NP — 266572.1; NC — 002662), PTS cellobiose-specific component IIC (accession), which has about 45% identity and its 13 to 470 amino acids were obtained from Clostridium acetobutylicum No .: NP — 34706.1; NC — 003030), PTS-based enzyme IIC component protein (Accession Number: NP — 2699944.1; NC — 002737), having about 42% identity and 17 to 468 amino acids obtained from Streptococcus pyogenes It has about 41% identity with a homologous protein, and its 17-468 amino acids are homologous to the PTS-based enzyme IIC component (accession number: NP_608069.1 |) (NC_003485) obtained from Streptococcus pyogenes It was shown to have about 41% identity with other proteins.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 30 (441 amino acids) has about 1 to 428 amino acids approximately 46 from a protein homologous to PTS cellobiose-specific enzyme IIC (accession number: NP — 472184.1; NC — 003212) obtained from Listeria innocure. About 46% of the protein having homology to PTS cellobiose-specific enzyme IIC (accession number: NP — 466230.1; NC — 003210) obtained from Listeria monocytogenes. The amino acids 10 to 427 have about 39% identity with a protein homologous to the PTS IIC component (Accession No .: NP_607435.1; NC_003485) obtained from Streptococcus pyogenes , Part 1 28 amino acids are approximately 36% identical to cellobiose-specific PTS IIC component protein (EC 2.7. 1.69) (accession number: NP — 266330.1; NC — 002662) obtained from Lactococcus lactis subspecies lactis And 1 to 421 amino acids have 31% identity with a protein homologous to the cellobiose phosphotransferase enzyme IIC component (Accession No: NP_4666201; NC_003210) obtained from Listeria monocytogenes. It was shown to have.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 32 (626 amino acids) has about 1 to 532 amino acids and a phosphotransferase system (PTS) arbutin-like enzyme IIBC component protein (accession number: NP — 388701.1; NC — 000964) obtained from Bacillus subtilis. 2 to 530 amino acids with about 51% identity with PTS arbutin-like enzyme IIBC component protein (Accession No: NP_5611112.1; NC_003366) obtained from Clostridium perfringens The PTS protein EII (accession number: gb | AAB63014.2; U81185) obtained from Fusobacterium mortiferum and about 52 amino acids. 1 to 533 amino acids having about 51% identity with MalP protein (accession number: gb | AAK695555.1; AF299082) obtained from Clostridium acetobutylicum, 1-533 amino acids were shown to have 51% identity with the PTS arbutin-like IIBC component protein (Accession Number: NP — 347171.1; NC — 003030) obtained from Clostridium acetobutylicum.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 34 (663 amino acids) has a sucrose-specific PTS IIBC component protein (EC 2.7. 1.69) obtained from Lactococcus lactis subspecies lactis (accession number: NP — 2672877.1; NC — 002662), which has about 58% identity, 5 to 471 amino acids of which are obtained from Staphylococcus aureus subsp. Aureus. PTS-based sucrose phosphotransferase enzyme IIBC component having about 54% identity with a protein homologous to session number: NP — 3734.29.1; NC — 002745), 5 to 472 amino acids obtained from Bacillus halodurans With about 46% identity with 4 to 468 amino acids from Salmonella enterica subsp. Enterica serovar Typhi (accession number: NP — 244441.1; NC — 002570). The obtained PTS IIBC component (accession number: NP — 45709.1; NC — 003198) has about 39% identity with a protein homologous thereto, and 4 to 468 amino acids are obtained from Salmonella typhimurium. It was shown to have about 39% identity with the resulting protein homologous to the phosphotransferase system IIB component (accession number: NP — 461505.1; NC — 003197).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 36 (665 amino acids) has 1 to 661 amino acids having about 44% identity with the PTS protein obtained from Clostridium perfringens (accession number: NP_561500.1; NC_003366). 1 to 657 amino acids have about 46% identity with a protein homologous to fructose-specific enzyme II, PTS BC component (Accession No: NP — 269062.1; NC — 002737) obtained from Streptococcus pyogenes 1 to 657 amino acids are approximately 46% identical to a protein homologous to fructose-specific enzyme II, PTS BC component (accession number: NP — 607065.1; NC — 003485) obtained from Streptococcus pyogenes A fructose-specific PTS-based enzyme IIBC component protein (EC 2.7. 1.69) (accession number: NP — 267115.1), of which 1 to 657 amino acids were obtained from Lactococcus lactis subsp. Lactis; NC_002662), which is about 45% identical and whose 1 to 660 amino acids were obtained from Bacillus halodurans, PTS-based fructose-specific enzyme IIBC component protein (accession number: NP — 241694.1; NC — 002570) And 43% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 38 (334 amino acids) has a sucrose operon repressor (Scr operon regulatory protein) (accession number: NP — 359313.1; NC — 003098) obtained from Streptococcus pneumoniae with about 4 to 334 amino acids. And 4 to 334 amino acids have about 46% identity with the LacI family of sugar-binding transcriptional regulators (accession number: NP_346232.1; NC_003028) obtained from Streptococcus pneumoniae , A sucrose operon repressor (Scr operon regulatory protein) obtained from Pediococcus pentosaceus (accession number: sp | P43472 | SCRR) PEDPE) with about 35% identity, 10 to 334 amino acids are about 35% identical to the ribose operon transcriptional repressor (Accession Number: NP_2445494.1; NC_002570) obtained from Bacillus halodurans. It was also shown that its 10 to 332 amino acids have 35% identity with the sucrose operon repressor (accession number: NP_346162.1; NC_003028) obtained from Streptococcus pneumoniae.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 40 (415 amino acids) has about 50% identity between 3 and 415 amino acids with ABC transporter substrate binding protein obtained from Streptococcus pneumoniae (accession number: NP_359212.1; NC_003098). And 19 to 389 amino acids have about 27% identity with a sugar-binding protein (accession number: NP — 53538.1; NC — 003306) obtained from Agrobacterium tumefaciens, 11-396 amino acids have about 25% identity with ABC transporter glycobinding protein (Accession Number: NP_488817.1; NC_003272) obtained from Nostocsp. PCC7120, 76-353 of which The amino acid has about 26% identity with a protein homologous to a sugar transporting sugar-binding protein (accession number: emb | CAB95275.1; AL3599779) obtained from Streptomyces coelicolor; 1 to 324 amino acids are found to have about 26% identity with a protein homologous to the sugar ABC transporter, Periplasma sugar binding protein (Accession Number: NP_470104.1; NC_003212), obtained from Listeria Innocure. It was done.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 42 (294 amino acids) is approximately 56% of the ABC transporter transmembrane permease-sugar transporter (accession number: NP_359211.1; NC — 003098) whose 10-285 amino acids were obtained from Streptococcus pneumoniae. About 38% of a protein homologous to the sugar ABC transporter permease protein (accession number: NP_464293.1; NC_003210) obtained from Listeria monocytogenes. A protein having the same identity and whose 7-285 amino acids are homologous to the sugar ABC transporter permease protein (Accession No: NP — 470102.1; NC — 003212) obtained from Listeria innocure. Lactose transport system permease protein (LacF) (accession number: NP_440703.1), which has about 38% identity with the protein and whose 12-286 amino acids are obtained from Synechocystis sp. PCC6803; NC_000911) with about 36% identity, and its 11-281 amino acids were obtained from Xylella fastidiosa, ABC transporter sugar permease (accession number: NP_299972.1; NC_002488) It was shown to have 36% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 44 (285 amino acids) is approximately 59% of which 12 to 285 amino acids are ABC transporter transmembrane permease-sugar transport protein (accession number: NP_359210.1; NC_003098) obtained from Streptococcus pneumoniae. 30 to 281 amino acids having about 32% identity with a protein obtained from Agrobacterium tumefaciens (accession number: NP — 356672.1; NC — 003063), and 30 to 281 Amino acid has about 32% identity with ABC transporter, transmembrane protein (sugar) obtained from Agrobacterium tumefaciens (accession number: NP_5344455.1; NC_003305), 10 to 28 of which The amino acid has about 33% identity with a protein homologous to the sugar ABC transporter, permease protein (Accession Number: NP — 463711.1; NC — 003210), obtained from Listeria monocytogenes, and 13 It was shown that ˜281 amino acids have about 34% identity with a protein homologous to the sugar ABC transporter, permease protein (Accession Number: NP — 46954.1; NC — 003212), obtained from Listeria innocure.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 46 (430 amino acids) is approximately 36% identical to sucrose-6-phosphate hydrolase (accession number: NP_359209.1; NC_003098) in which 2 to 429 amino acids are obtained from Streptococcus pneumoniae. 2 to 429 amino acids having about 36% identity with a protein homologous to sucrose-6-phosphate hydrolase (Accession Number: NP_34628.1; NC_003028) obtained from Streptococcus pneumoniae, Its 18-373 amino acids have about 36% identity with β-fructosidase (accession number: NP — 2292155.1; NC — 000853) obtained from Thermotoga maritima, and the 21 to 405 amino acids are ,The It has about 31% identity with β-fracturanosidase (EC 3.2.1.26) (accession number: pi || JU0460) obtained from Zymomonas mobilis 21-362 amino acids were shown to have 35% identity with sucrose-6 phosphate hydrolase (accession number: NP — 311270.1; NC — 002695) obtained from E. coli.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 48 (368 amino acids) is approximately 65 to 366 amino acids in which multiple sugar-binding transport ATP-binding protein (msmK) (accession number: sp | Q00752 | MSMK_STRMU) obtained from Streptococcus mutans is obtained. % -Identity of which 1 to 366 amino acids are approximately 65% identical to the multiple sugar-binding ABC transport system (ATP binding protein) obtained from Streptococcus pyogenes (accession number: NP — 269942.1; NC — 002737) ABC transporter ATP-binding protein-multiple sugar transport (accession number: NP — 359030.1; NC — 0030), of which 1 to 367 amino acids were obtained from Streptococcus pneumoniae 98) with about 66% identity, the amino acids 1 to 366 of which are obtained from Streptococcus pyogenes, a multiple sugar-binding ABC transport system (ATP-binding protein) (accession number: NP — 608016.1; NC — 003485) It has about 65% identity, and its amino acids 1 to 367 are 66% identical to the sugar ABC transporter, ATP binding protein (accession number: NP — 346026.1; NC — 003028) obtained from Streptococcus pneumoniae It was shown to have sex.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 50 (490 amino acids) has about 63% identity between its 11 to 489 amino acids and the gtfA protein (accession number: pi || BWSOGM) obtained from Streptococcus mutans, 11-490 amino acids have about 63% identity with sucrose phosphoridase (EC 2.41.7) (accession number: pi || A27626) obtained from Streptococcus mutans, 11 ˜489 amino acids have about 63% identity with sucrose phosphoridase (sucrose glucosyltransferase) (accession number: sp | P10249 | SUCP_STRMU) obtained from Streptococcus mutans, , Streptococcus It has about 63% identity with dextransucrase (sucrose 6-glucosyltransferase) (accession number: NP — 359301.1; NC — 003098), obtained from S. pneumoniae, whose 11-484 amino acids were obtained from Streptococcus pneumoniae, It was shown to have 63% identity with sucrose phosphorylase (accession number: NP — 346255.1; NC — 003028).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 52 (328 amino acids) is approximately 55% of the 47-316 amino acids with ribose ABC transporter (ribose binding protein) obtained from Bacillus subtilis (accession number: NP_3917477.1; NC_000964). 5 to 323 amino acids having about 45% identity with the ribose ABC transporter substrate binding protein (accession number: NP — 267791.1; NC — 002662) obtained from Lactococcus lactis subspecies lactis 4 to 278 amino acids, approximately 42% identical to ribose binding protein (accession number: dbj | BAA31869.1; AB009593) obtained from Tetragenococcus halophilus 15 to 316 amino acids have approximately 39% identity with ribose ABC transporter (ribose binding protein) (accession number: NP_244599.1; NC_002570) obtained from Bacillus halodurans It was also shown that the 4-315 amino acids have 42% identity with the RbsB protein (Accession number: NP — 245090.1; NC — 002663) obtained from Pasteurella multocida.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 54 (285 amino acids) is approximately 60% identical to ribose ABC transporter (permease) (accession number: NP_391476.1; NC_000964) obtained from Bacillus subtilis in 1 to 277 amino acids. 1 to 277 amino acids have about 59% identity with ribose transport system permease protein (rbcS) (accession number: sp | P36948 | RBSC_BASU) obtained from Bacillus subtilis, 4 to 277 amino acids have about 57% identity with the ribose ABC transporter (permease) (Accession number: NP_244598.1; NC_002570) obtained from Bacillus halodurans, Lactococca It has about 58% identity with the ribose ABC transporter permease protein (accession number: NP — 267792.1; NC — 002662) obtained from Su. Lactis subsp. Lactis, and its 4-278 amino acids are It was shown to have 54% identity with the D-ribose ABC transporter, permease protein (rbsC) (accession number: NP_438661.1; NC_000907), obtained from Haemophilus influenzae.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 56 (496 amino acids) is approximately 59% of which 5 to 496 amino acids are ribose ABC transporter ATP-binding protein obtained from Lactococcus lactis subspecies lactis (accession number: NP — 26793.1; NC — 002662). And 5 to 496 amino acids have about 57% identity with ribose ABC transporter (ATP binding protein) (accession number: NP_391475.1; NC_000964) obtained from Bacillus subtilis. And 5 to 496 amino acids have about 51% identity with the ATP binding protein (accession number: pi || 14065) obtained from Bacillus subtilis, and 5 to 495 amino acids are Bacillus halo. Obtained from Durance, Ribose ABC transporter (ATP binding protein) (accession number: NP_244597.1; NC_002570) has about 49% identity, and its 7-494 amino acids were obtained from Agrobacterium tumefaciens , ABC transporter, nucleotide binding / ATPase protein [ribose] (accession number: NP_5333484.1; NC_003304), was shown to have 45% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 58 (134 amino acids) is about 4 to 134 amino acids from ribose permease (RbsD) (accession number: gb | AAD34337.1; AF115391) obtained from Lactobacillus sakei. About 51% identity with a protein having 58% identity and whose 4-134 amino acids are homologous to the ribose ABC transporter (Accession Number: NP — 52547.1; NC — 00366) obtained from Clostridium perfringens The 4-132 amino acids have about 50% identity with the ribose ABC transporter permease protein (Accession Number: NP_267794.1; NC_002662) obtained from Lactococcus lactis subsp. Lactis 4 to 134 amino acids have about 45% identity with ribose ABC transporter (permease) (accession number: NP_244596.1; NC_002570) obtained from Bacillus halodurans. 134 amino acids were shown to have 51% identity with ribose permease (accession number: NP — 370793.1; NC — 002758) obtained from Staphylococcus aureus subspecies aureus.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 60 (308 amino acids) is about 51% identical in that 4-301 amino acids are ribokinase (RbsK) obtained from Lactobacillus sakei (accession number: gb | AAD3433381; AF115391). 1 to 303 of which have about 48% identity with a protein homologous to ribokinase (accession number: NP_370792.1; NC_002758) obtained from Staphylococcus aureus subspecies aureus 3 to 305 amino acids have about 45% identity with ribokinase (accession number: NP — 52548.1; NC — 00366) obtained from Clostridium perfringens, and 1 to 299 amino acids are hemophilus Ribokina from flu Ribokinase (accession number: NP_4033674.1), which has about 41% identity with RbsK (accession number: NP — 43863.1; NC — 000907), whose 2-300 amino acids were obtained from Yersinia pestis. NC_003143) and 38% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 62 (285 amino acids) is approximately 63% of which 1 to 285 amino acids are maltose ABC transporter permease protein obtained from Lactococcus lactis subspecies lactis (accession number: NP — 267841.1; NC — 002662). About 6 to 284 amino acids of which the maltose / maltodextrin ABC transport system protein (permease) obtained from Streptococcus pyogenes (accession number: NP — 26943.1; NC — 002737) is approximately 54. % Homologous protein whose 12-284 amino acids are homologous to the malG protein (accession number: pi || S63616) obtained from Klebsiella oxytoca About 39% of the maltose / maltodextrin transport system (permease) (accession number: NP — 243790.1; NC — 002570) with about 38% identity, 9-285 amino acids obtained from Bacillus halodurans It has about 36% identity with a protein having homology with a maltodextrin transport system permease (accession number: NP_391294.1; NC_000964) obtained from Bacillus subtilis. It was shown to have.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 64 (452 amino acids) is approximately 63% of which 1 to 452 amino acids are maltose ABC transporter permease protein obtained from Lactococcus lactis subspecies lactis (accession number: NP — 267840.1; NC — 002662). The amino acids 3 to 452 are about 52 with a protein homologous to maltose / maltodextrin ABC transport system protein (permease) (accession number: NP — 2694222.1; NC — 002737) obtained from Streptococcus pyogenes. % Maltose / maltodextrin ABC transport system (permease), whose amino acids 3 to 452 were obtained from Streptococcus pyogenes (accession number: NP_6074 2.1; NC_003485) and a protein homologous to the malF protein (Accession No. pir || S63615) having about 52% identity with 28 to 451 amino acids from Klebsiella oxytoca And about 23% of its amino acids 23-451 with maltose / maltodextrin transport system permease (accession number: NP_243791.1; NC_002570) obtained from Bacillus halodurans. It was shown to have identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 66 (408 amino acids) is approximately 49% of which 1 to 407 amino acids are maltose ABC transporter substrate binding protein obtained from Lactococcus lactis subspecies lactis (accession number: NP — 267839.1; NC — 002662). About 37% identity with a protein homologous to the maltose / maltodextrin-binding protein (accession number: NP — 607421.1; NC — 003485) obtained from Streptococcus pyogenes. Of which 1 to 405 amino acids are homologous to maltose / maltodextrin-binding protein (accession number: NP — 26921.1; NC — 002737) obtained from Streptococcus pyogenes It has about 36% identity with the protein, and its 1 to 393 amino acids are homologous to maltose / maltodextrin ABC transporter (binding protein) (accession number: NP_471563.1; NC_003212) obtained from Listeria innocure About 1 to 403 amino acids and homologous to a maltose / maltodextrin-binding protein (accession number: NP_391296.1; NC_000964) obtained from Bacillus subtilis. It was shown to have about 26% identity with the protein.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 68 (368 amino acids) is approximately 64% of the polysaccharide binding transport ATP-binding protein (msmK) (accession number: sp | Q00752 | MSMK_STRMU) obtained from Streptococcus mutans. 1 to 366 amino acids, and 1 to 366 amino acids of which were obtained from Streptococcus pyogenes, a polysaccharide-binding ABC transport system (ATP-binding) protein (accession number: NP — 269942.1; NC — 002737). About 64% identity with about 1 to 366 amino acids from a polysaccharide-bound ABC transport system (ATP-bound) protein (Accession Number: NP — 608016.1; NC — 003485) obtained from Streptococcus pyogenes Same 1 to 366 amino acids having about 64% identity with ABC transporter ATP-binding protein-polysaccharide transport (accession number: NP — 359030.1; NC — 003098) obtained from Streptococcus pneumoniae, and The polysaccharide ABC transporter ATP binding protein (accession number: NP — 266777.1; NC — 002662) obtained from Lactococcus lactis subspecies lactis was shown to have 63% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 70 (512 amino acids) has about 1 to 510 amino acids approximately identical to a protein homologous to the sugar ABC transporter (ATP binding protein) obtained from Streptococcus pyogenes (accession number: NP_2699365.1; NC_002737). About 60% identity with a protein homologous to the sugar ABC transporter (ATP-binding protein) (accession number: NP_6072966.1; NC_003485) obtained from Streptococcus pyogenes. % Sugar identities, 5 to 503 amino acids of which were obtained from Lactococcus lactis subspecies lactis, sugar ABC transporter ATP binding protein (accession number: NP — 267484.1; NC — 00 662) and about 61% of the sugar ABC transporter, ATP-binding protein (accession number: NP — 3453377.1; NC — 003028) obtained from Streptococcus pneumoniae. And whose amino acids 7 to 503 are 60% of the ABC transporter ATP-binding protein-ribose / galactose transport protein (accession number: NP_358342.1; NC_003098) obtained from Streptococcus pneumoniae It was shown to have identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 72 (383 amino acids) is approximately the same as the sugar ABC transporter permease protein (accession number s. NP — 2674855.1; NC — 002662), whose 7 to 351 amino acids were obtained from Lactococcus lactis subspecies lactis. About 47% of the ABC transporter transmembrane permease (ribose / galactose transport) (accession number: NP — 358343.1; NC — 003098) with 49% identity and 4 to 351 amino acids obtained from Streptococcus pneumoniae The sugar ABC transporter, permease protein (accession number: NP — 3453388.1; NC — 003), whose amino acids 4 to 351 were obtained from Streptococcus pneumoniae 048), a sugar ABC transporter (permease protein) (accession number: NP — 269364.1; NC — 002737), which has about 47% identity with a protein homologous to that of 028) and whose 4-342 amino acids are obtained from Streptococcus pyogenes ), And a sugar ABC transporter (permease protein) (accession number: NP — 607295.1; 4 to 342 amino acids obtained from Streptococcus pyogenes). It was shown to have about 49% identity with a protein homologous to NC_003485).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 74 (318 amino acids) is a protein whose 1 to 318 amino acids are homologous to a sugar ABC transporter (permease protein) (accession number: NP — 6072944.1; NC — 003485) obtained from Streptococcus pyogenes. About 67% identity, about 1 to 318 amino acids from a protein homologous to the sugar ABC transporter (permease protein) obtained from Streptococcus pyogenes (accession number: NP_2699363.1; NC_002737) Glucose ABC transporter, permease protein (accession number: NP — 34539.1; NC — 0) with 66% identity, from 1 to 318 amino acids obtained from Streptococcus pneumoniae 3028), a sugar ABC transporter permease protein (accession number: NP — 2674866.1), which has about 65% identity and whose 1 to 318 amino acids were obtained from Lactococcus lactis subsp. Lactis NC_002662) with about 63% identity, and its 6-318 amino acids are homologous to the sugar ABC transporter (permease protein) obtained from Listeria Inocure (accession number: NP_470764.1; NC_003212) It was shown to have about 61% identity with other proteins.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 76 (450 amino acids) is a protein whose 11-448 amino acids are homologous to a sugar transporter (accession number: NP — 273437.1; NC — 003112) obtained from Neisseria meningitidis. It has about 68% identity, and its 11 to 448 amino acids are about the same as a protein homologous to the integral membrane transport protein (accession number: NP — 28477.17.1; NC — 003116) obtained from Neisseria meningitidis. About 39% identity with a protein that has 68% identity and whose 17-229 amino acids are homologous to the transporter (accession number: NP — 421086.1; NC — 002696) obtained from Caulobacter crescentus And its 31-450 amino acids have about 21% identity with a sucrose transporter (accession number: gb | AAG09270.1; AF176950) obtained from Lycopersicon esculentum, and 31-442 amino acids were shown to have 21% identity with a sucrose transporter (accession number gb | AAG09191.1; AF175321) obtained from Arabidopsis thaliana.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 78 (495 amino acids) has about 32% identity between its 8-482 amino acids and the transporter protein obtained from Lactococcus lactis subspecies lactis (accession number: NP — 266394.1; NC — 002662). 8 to 482 amino acids have about 34% identity with a protein homologous to the efflux transporter (Accession Number: NP — 464506.1; NC — 003210) obtained from Listeria monocytogenes, ˜482 amino acids have approximately 34% identity with a protein homologous to the efflux transporter (Accession Number: NP — 470317.1; NC — 003212) obtained from Listeria Inocure, the 7 to 422 amino acids are Clostridial Ace Membrane transport with about 30% identity with MDR-related permease (accession number: NP — 1492944.1; NC — 001988) obtained from butyricum, and its 8-425 amino acids were obtained from Streptomyces coelicolor It was shown to have about 29% identity with a protein homologous to the protein (accession number: emb | CAB89031.1; AL353870).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 80 (471 amino acids) has about 32% identity between 1 and 440 amino acids with a transport protein obtained from Lactococcus lactis subspecies lactis (accession number: NP — 266394.1; NC — 002662). 1 to 464 amino acids have about 34% identity with a protein homologous to the efflux transporter (accession number: NP — 464506.1; NC — 003210) obtained from Listeria monocytogenes. 464 amino acids have about 34% identity with a protein homologous to the efflux transporter (Accession No .: NP — 470317.1; NC — 003212) obtained from Listeria innocure, whose 1 to 412 amino acids are Clostridium acetobutylicum An exporter having about 29% identity with the MDR-related permease (accession number: NP — 1492944.1; NC — 001988) obtained from the same, and 4 to 459 amino acids obtained from Streptomyces coelicolor. Accession number: pi || T36377) and was shown to have about 28% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 82 (412 amino acids) is approximately 49% identical to a protein whose 18-400 amino acids are homologous to a drug efflux transporter obtained from Listeria Inocure (accession number: NP — 47222.1; NC — 003212). Having about 49% identity with a protein homologous to a drug efflux transporter (accession number: NP — 466263.1; NC — 003210) obtained from Listeria monocytogenes, Its 18 to 397 amino acids have about 48% identity with a protein homologous to a transport protein obtained from E. coli (accession number: NP — 415571.1; NC — 000913), and its 15 to 399 amino acids are Lactococcus lactis Subspecies Rakute The protein (Accession number: NP — 2662822.1; NC — 002662), which is a multi-drug resistant excretion pump obtained from S. cerevisiae, has about 47% identity, and its 18-399 amino acids were obtained from Salmonella typhimurium. It was shown to have about 48% identity with proteins homologous to the MFS family transporter protein (accession number: NP_460125.1; NC_003197).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 84 (462 amino acids) is about 38% identical in its 9-413 amino acids to ORFC (accession number: emb | CAB61253.1; AJ250422) obtained from Oenococcus oeni. 2 to 378 amino acids have about 38% identity with the transporter protein (Accession No: NP — 26695.1; NC — 002662) obtained from Lactococcus lactis subspecies lactis, and 6 to 411 amino acids thereof. Has approximately 34% identity to a protein homologous to a drug resistance protein obtained from Streptococcus pyogenes (accession number: Np — 606824.1; nc — 003485), and its 6 to 411 amino acids were obtained from Streptococcus pyogenes Drug excretion with about 33% identity to a protein homologous to a drug resistant protein (accession number: NP — 268834.1; NC — 002737), with 2 to 454 amino acids from Lactococcus lactis subspecies lactis It was shown to have 34% identity with the protein (accession number: NP_2675404.1; NC_002662).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 86 (490 amino acids) is about 55% identical to a protein having 3 to 476 amino acids homologous to a drug efflux protein obtained from Listeria monocytogenes (accession number: NP_466111.1; NC_003210). 3 to 476 amino acids have about 54% identity with a protein homologous to a drug efflux protein obtained from Listeria Inocure (accession number: NP — 472062.1; NC — 003212). 478 amino acids have about 45% identity with the multidrug resistance protein from Lactococcus lactis subsp. Lactis (Accession Number: NP — 27065.1; NC — 002662), 8 to 484 amino acids of which are Bacillus subtilis Multi-drug resistant tape obtained from A multidrug resistant protein (accession number: accession number: NP_3888266.1; NC_000964) having about 49% identity with 18 to 425 amino acids from Bacillus subtilis. NP — 388782.1; NC — 000964) and was shown to have about 44% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 88 (416 amino acids) is approximately 26% of which 17 to 408 amino acids are a protein obtained from Desulfitobacterium hafniense (accession number: gb | AAL877781.1; AF403184). Of which 26-408 amino acids have about 25% identity with the major facilitator superfamily transporter (accession number: NP_359046.1; NC_003098) obtained from Streptococcus pneumoniae. And its 61-399 amino acids have about 21% identity with a protein homologous to the efflux protein obtained from Campylobacter jejuni (accession number: NP_282813.1; NC_002163), 5 to 368 amino acids have about 19% identity with a protein homologous to MFS permease (accession number: NP_533033.1; NC_003304) obtained from Agrobacterium tumefaciens, 205 amino acids were shown to have 25% identity with a multidrug resistance protein (Accession Number: NP — 244175.1; NC — 002570) obtained from Bacillus halodurans.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 90 (548 amino acids) is approximately 38% identical to a protein whose 17-546 amino acids are homologous to a transporter protein (Accession No .: NP — 471001.1; NC — 003212) obtained from Listeria innocure. Having 17 to 546 amino acids having about 37% identity with a protein homologous to a transporter protein obtained from Listeria monocytogenes (accession number: NP — 465149.1; NC — 003210); 534 amino acids have about 36% identity with the polysaccharide transporter protein obtained from Streptococcus pneumoniae (accession number: NP — 358976.1; NC — 003098), 17 to 534 amino acids, It has about 36% identity with a protein homologous to a polysaccharide biosynthetic protein obtained from T. pneumoniae (accession number: NP_345978.1; NC_003028), and its 12-546 amino acids are lactococcus It was shown to have 35% identity with a hypothetical protein obtained from lactis subspecies lactis (accession number: NP_267962.1; NC_002662).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 92 (485 amino acids) is approximately 44% of a protein having 1 to 484 amino acids homologous to the efflux transporter protein obtained from Listeria monocytogenes (accession number: NP — 464506.1; NC — 003210). 1 to 484 amino acids having about 44% identity with a protein homologous to the efflux transporter protein obtained from Listeria Inocure (accession number: NP — 470317.1; NC — 003212), 9-420 amino acids have about 34% identity to the MDR-related permease protein (Accession Number: NP — 1492944.1; NC — 001988), obtained from Clostridium acetobutylicum, whose 12-475 amino acids are It has about 33% identity with the transporter protein (Accession number: NP — 266394.1; NC — 002662) obtained from Tococcus lactis subsp. ) From the hypothetical protein obtained from (accession number: emb | CAB37973.1; X76640).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 94 (199 amino acids) is approximately 46% identical to a protein whose 23-173 amino acids are homologous to a drug efflux transporter protein (Accession No: NP — 47222.12.1; NC — 003212) obtained from Listeria innocure And its amino acids 23 to 173 have about 45% identity with a protein homologous to the drug efflux transporter protein obtained from Listeria monocytogenes (accession number: NP_4666263.1; NC_003210). , Whose amino acids 23-173 have about 49% identity with the multidrug resistance efflux pump protein (Accession number: NP — 266282.1; NC — 002662) obtained from Lactococcus lactis subsp. Lactis, 17 The amino acid has about 46% identity with a protein homologous to the efflux pump protein obtained from Salmonella enterica subsp. Enterica cellovar typhi (accession number: NP — 45497.1; NC — 003198), It was shown that 173 amino acids have about 46% identity with a protein homologous to the permease obtained from Salmonella typhimurium (accession number: NP — 49577.1; NC — 003197).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 96 (538 amino acids) has 4 to 525 amino acids having about 32% identity with a polysaccharide transporter obtained from Streptococcus pneumoniae (accession number: NP — 358976.1; NC — 003098), 5 to 525 amino acids have about 32% identity with a protein homologous to the polysaccharide biosynthetic protein obtained from Streptococcus pneumoniae (accession number: NP_345978.1; NC_003028), and the 5 to 526 amino acids are , Approximately 33% identity with a conserved virtual protein (Accession Number: NP — 606680.1; NC — 003485) obtained from Streptococcus pyogenes, of which 5 to 526 amino acids are Streptococcus A virtual protein obtained from Piognes, having approximately 33% identity with a conserved virtual protein (Accession Number: NP — 268708.1; NC — 002737), and obtained from Lactococcus lactis subspecies lactis (Accession Number: NP_267962.1; NC_002662) and 30% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 98 (328 amino acids) is approximately 57% of the sucrose operon regulatory protein (scrR) (accession number: sp | P43472 | SCRR_PEDPE) whose 1 to 323 amino acids are obtained from Pediococcus pentosaceus. 1 to 322 amino acids having about 51% identity with the sucrose operon repressor (Accession Number: NP — 346162.1; NC — 003028) obtained from Streptococcus pneumoniae. The amino acid has about 49% identity with the sucrose operon regulatory protein (scrR) (accession number: sp | Q54430 | SCRR_STRMU) obtained from Streptococcus mutans, It has about 49% identity with a protein homologous to the sucrose operon repressor (accession number: NP — 607889.1; NC — 003485) obtained from Leptococcus pyogenes, and 1 to 322 amino acids thereof are Streptococcus pyogenes. And was found to have 49% identity with a protein homologous to the sucrose operon repressor (accession number: Np — 269821.1; nc — 002737).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 100 (485 amino acids) has sucrose-6-phosphate hydrolase (ScrB) (accession number: pi || S68598) in which 1 to 466 amino acids are obtained from Streptococcus sobrinus. It has about 50% identity, and its 1 to 461 amino acids have about 49% identity with sucrose-6-phosphate hydrolase (accession number: NP_359160.1; NC_003098) obtained from Streptococcus pneumoniae. 1 to 461 amino acids having about 49% identity with sucrose-6-phosphate hydrolase (Accession No: NP — 346161.1; NC — 003028) obtained from Streptococcus pneumoniae Amino acids It has about 49% identity with a protein homologous to sucrose-6-phosphate hydrolase (accession number: NP — 607888.1; NC — 003485) obtained from Streptococcus pyogenes, and its 1 to 466 amino acids are Streptococcus -It was shown to have 49% identity with a protein homologous to sucrose-6-phosphate hydrolase obtained from Piogenes (accession number: NP — 269820.1; NC — 002737).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 102 (649 amino acids) has a phosphotransferase enzyme II (EC 2.7.1.69), a sucrose-specific IIABC component protein (accession) in which 1 to 645 amino acids are obtained from Streptococcus mutans. No .: sp | P12655 | PTSA_STRMU), about 1 to 647 amino acids obtained from Pediococcus pentosaceus, phosphotransferase enzyme II (EC 2.7.1.69, sucrose) Specific enzyme IIABC (accession number: sp | P43470 | PTSA_PEDPE) Enzyme II sucrose protein (accession number: emb), which has about 56% identity and 1 to 643 amino acids obtained from Lactococcus lactis ｜ C AB09690.1; Z97015), 114-647 amino acids of which are obtained from Lactobacillus sakei, PTS sucrose-specific enzyme II (accession number: gb | AAK92528.1; Phosphotransferase system IIB component protein (accession number: NP_601842.1), which has about 52% identity with AF401046) and whose 1-621 amino acids were obtained from Corynebacterium glutamicum; NC_003450) and 45% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 104 (667 amino acids) is a β-glucoside-specific PTS IIABC component protein (EC 2.7. 1.69) whose 192-661 amino acids were obtained from Lactococcus lactis subsp. Session number: NP — 266583.1; NC — 002662), phosphotransferase system (PTS) β-glucoside specific enzyme IIABC (191 to 652 amino acids obtained from Listeria monocytogenes) PTS-dependent enzyme II (accession number: accession number: NP — 464560.1; NC — 003210), which has about 39% identity and whose 191 to 662 amino acids were obtained from Clostridium longisporum. gb | A AC05713.1; L49336), PTS β-glucoside-specific enzyme II ABC component protein (accession number: NP — 241461) having about 37% identity, whose 191 to 666 amino acids were obtained from Bacillus halodurans. .1; NC_002570) and having about 19% identity, and its 191-650 amino acids were obtained from Listeria Inocure, PTS system, β-glucoside specific enzyme IIABC protein (Accession No .: NP — 469373. 1; about 36% identity with a protein homologous to NC — 003212).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 106 (241 amino acids) has about 47% identity between 1 and 238 amino acids with the trehalose operon transcription repressor (accession number: sp | P39796 | TRER_BACSU) obtained from Bacillus subtilis. 4 to 238 amino acids have about 41% identity with the transcriptional repressor of trehalose operon obtained from Bacillus halodurans (accession number: NP — 241739.1; NC — 002570), and 9 to 237 amino acids are , Having about 44% identity with a protein homologous to the transcriptional regulator GntR family (Accession number: NP — 4705588.1; NC — 003212) obtained from Listeria innocure, and 9 to 237 amino acids thereof are Listeria monocytogenes. Obtained from the GntR family of transcriptional regulators (accession number: NP — 4647878.1; NC — 003210) and having about 44% identity, the 5-238 amino acids are derived from Lactococcus lactis subspecies lactis. The obtained GntR family transcriptional regulator (accession number: NP — 266581.1; NC — 002662) was shown to have 41% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 108 (570 amino acids) has about 56% identity with a protein in which 22 to 566 amino acids are homologous to dextran glucosidase obtained from Streptococcus pyogenes (accession number: NP_608103.1; NC_003485). The amino acids 23 to 568 have about 57% identity with dextran glucosidase obtained from Streptococcus pneumoniae (accession number: NP — 359290.1; NC — 003098), and the amino acids 22 to 566 are from Streptococcus pyogenes. It has about 56% identity with a protein homologous to the obtained dextran glucosidase (accession number: NP — 26006.1; NC — 002737), and its 23 to 568 amino acids are It has about 57% identity with a protein homologous to dextran glucosidase DexS (accession number: NP — 346315.1; NC — 003028) obtained from Leptococcus pneumoniae, and its 17 to 570 amino acids are Clostridium perfringen It was shown to have 54% identity with α-glucosidase (accession number: NP — 51478.1; NC — 00366) obtained from

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 110 (370 amino acids) has about 1 to 368 amino acids that are about 67% of the ABC transporter ATP-binding protein-polysaccharide transport protein (accession number: NP — 359030.1; NC — 003098) obtained from Streptococcus pneumoniae. 1 to 368 amino acids have about 67% identity with the sugar ABC transporter, ATP-binding protein (Accession No: NP — 346026.1; NC — 003028), obtained from Streptococcus pneumoniae, Its 1 to 368 amino acids are approximately 66% identical to the polysaccharide binding transport ATP binding protein (msmK) (accession number: sp | Q00752 | MSMK_STRMU) obtained from Streptococcus mutans 1 to 365 amino acids have about 68% identity with a protein homologous to the sugar ABC transporter, ATP-binding protein (Accession No .: NP — 469649.1; NC — 003212), obtained from Listeria Inocure In addition, the amino acids 1 to 365 thereof have 67% identity with a protein homologous to the sugar ABC transporter, ATP-binding protein (Accession No .: NP_4633809.1; NC_003210) obtained from Listeria monocytogenes. It was shown that.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 112 (278 amino acids) is approximately 81% in which 2-278 amino acids are polysaccharide binding transport system permease protein (msmG) (accession number: sp | Q00751 | MSMG_STRMU) obtained from Streptococcus mutans. 1 to 278 amino acids have 73% identity with the sugar ABC transporter, permease protein (Accession Number: NP — 346266.1; NC — 003028), obtained from Streptococcus pneumoniae, Its 2-278 amino acids are approximately 72% identical to ABC transporter transmembrane permease-multiple sugar (Accession Number: NP — 359302.1; NC — 003098) obtained from Streptococcus pneumoniae Of which the amino acids 72 to 278 have about 85% identity with the hypothetical protein fragment obtained from Streptococcus mutans (accession number: pi || B27626), and the amino acids 4 to 278 are It was shown to have 44% identity with the sugar permease (accession number: NP_350251.1; NC_003030), obtained from Clostridium acetobutylicum.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 114 (291 amino acids) is approximately 73% of the ABC transporter transmembrane permease-multiple sugar (accession number: NP — 359303.1; NC — 003098) obtained from Streptococcus pneumoniae. 4 to 290 amino acids having about 73% identity with the sugar ABC transporter, permease protein (accession number: NP_346327.1; NC_003028) obtained from Streptococcus pneumoniae, Multiple sugar-binding permease protein (msmF) (accession number: sp | Q00750 | MSMF_STRMU) whose 1-290 amino acids were obtained from Streptococcus mutans About 53% with ABC type sugar transport system, permease component protein (accession number: NP_350252.1; NC_003030) obtained from Clostridium acetobutylicum. In which the potential amylolytic product transport system permease protein (accession number: sp |) was obtained from Thermoanaerobacterium thermosulfurigenes, whose 2-291 amino acids were obtained from Thermoanaerobacterium thermosulfurigenes. P37730 | AMYD_THETU) and 32% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 116 (423 amino acids) is approximately 56% identical to a multiple sugar binding protein precursor (accession number: sp | Q00749 | MSME_STRMU) in which 8 to 421 amino acids are obtained from Streptococcus mutans. 9 to 421 amino acids have about 56% identity with the sugar ABC transporter, sugar-binding protein (accession number: NP_346328.1; NC_003028) obtained from Streptococcus pneumoniae, ~ 421 amino acids have about 56% identity with ABC transporter substrate binding protein-multiple sugar (Accession Number: NP_359304.1; NC_003098) obtained from Streptococcus pneumoniae 9-420 amino acids have about 29% identity with ABC-type sugar transport system, Periplasma sugar-binding component protein (accession number: NP_350253.1; NC_003030), obtained from Clostridium acetobutylicum, and It has been shown that 6-412 amino acids have approximately 24% identity with a protein homologous to multiple sugar binding protein (Accession Number: NP_391140.1; NC_000964) obtained from Bacillus subtilis.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 118 (279 amino acids) is approximately 57% of which 1 to 273 amino acids are raffinose operon transcription regulatory protein (rafR) (accession number: sp | P43465 | RAFR_PEDPE) obtained from Pediococcus pentosaceus. The 5-273 amino acids have about 35% identity with a protein homologous to the transcriptional regulatory factor (msmR) obtained from Streptococcus mutans (accession number: pi || A42400). And its 5-273 amino acids have about 35% identity with the msm operon regulatory protein (accession number: sp | Q00753 | MSMR_STRMU) obtained from Streptococcus mutans, Streptococcus The msm operon regulatory protein (accession number: NP — 466330.1; NC — 003028) obtained from Pneumoniae has about 36% identity, and its 19-273 amino acids are obtained from Streptococcus pneumoniae. Sugar metabolism) was shown to have 36% identity with the operon regulatory protein (accession number: NP_359306.1; NC_003098).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 120 (277 amino acids) is approximately 28% of a protein whose 37-141 amino acids are homologous to rRNA methylase (accession number: NP — 218549.1; NC — 000919) obtained from Treponema pallidum. The amino acids 74-141 have about 32% identity with GTP-binding nucleoprotein RAN (accession number: NP_113408.1; NC_002753) obtained from Guillardia theta. 75-141 amino acids are about 29% identical to GTP-binding nucleoprotein RAN / TC4 (accession number: sp | P33519 | RAN_DICDI) obtained from Dictyostelium discoideum. Have Furthermore, it was shown that 140 to 190 amino acids have about 25% identity with a hypothetical protein obtained from Arabidopsis thaliana (accession number: NP — 191798.1; NM — 116104).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 122 (530 amino acids) has an ABC transporter ATP binding / permease protein (Accession Number: NP — 2676788.1; NC — 002662), 8 to 524 amino acids obtained from Lactococcus lactis subspecies lactis It has about 26% identity, and its 49-518 amino acids have about 25% identity with the ABC transporter, ATP-binding protein (accession number: NP_344680.1; NC_003028) obtained from Streptococcus pneumoniae. The ABC transporter ATP binding / transmembrane permease (accession number: NP — 357731.1; NC — 003098) obtained from Streptococcus pneumoniae 25% identity, 47-511 amino acids have about 24% identity with ABC transporter (accession number: NP — 440626.1; NC — 000911) obtained from Synechocystis sp. PCC6803, and Its 7-511 amino acids were shown to have about 24% identity with proteins homologous to the ABC transporter (ATP binding protein) obtained from Bacillus subtilis (accession number: NP — 388852.1; NC — 000964). .

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 124 (530 amino acids) has an ABC transporter ATP binding / permease protein (accession number: NP — 2676788.1; NC — 002662), whose 4 to 524 amino acids are obtained from Lactococcus lactis subspecies lactis It has about 24% identity, and its 55-508 amino acids have about 25% identity with the ABC transporter, ATP binding protein (Accession Number: NP_344680.1; NC_003028) obtained from Streptococcus pneumoniae. The ABC transporter ATP binding / transmembrane permease (accession number: NP — 357731.1; NC — 003098) obtained from Streptococcus pneumoniae About 24% identity with the drug efflux ABC transporter, ATP binding / permease (accession number: NP — 345800.1; NC — 003028), which has 25% identity and whose amino acids 1 to 511 were obtained from Streptococcus pneumoniae And 1 to 511 amino acids have 24% identity with ABC transporter ATP binding / transmembrane protein (Accession No .: NP_358796.1; NC_003098) obtained from Streptococcus pneumoniae It has been shown.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 126 (527 amino acids) is an ABC transporter ATP binding / permease protein (accession number: NP — 2676788.1; NC — 002662), 8 to 527 amino acids obtained from Lactococcus lactis subspecies lactis About 24% with ABC transporter ATP binding / transmembrane permease protein (accession number: NP_3577731.1; NC_003098) having about 25% identity, 13 to 520 amino acids obtained from Streptococcus pneumoniae The ABC transporter, ATP binding protein (accession number: NP — 344680.1; NC — 003), whose 13-520 amino acids were obtained from Streptococcus pneumoniae 28) a drug efflux ABC transporter, ATP-binding / permease protein (accession number: NP — 345800.1; NC — 003028), having about 24% identity with 22 to 511 amino acids from Streptococcus pneumoniae About 22% identity, 22-511 amino acids of which are 22% identical to ABC transporter ATP binding / transmembrane protein (accession number: NP_358796.1; NC_003098) obtained from Streptococcus pneumoniae It was shown to have sex.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 128 (534 amino acids) has about 23% identity between its 14-512 amino acids and the comA protein obtained from Streptococcus pneumoniae (accession number: pi || A39203). 512 amino acids have about 26% identity with Lactococcin A transport ATP binding protein (lcnC) (accession number: sp | Q00564 | LCNC_LACLA), obtained from Lactococcus lactis, 512 amino acids have about 23% identity with the transporter ATP binding protein (ComA) (Accession number: NP — 357637.1; NC — 003098) obtained from Streptococcus pneumoniae, 113 to 509 amino acids are About 25% identity with ABC transporter (Accession number: gb | AAC72026.1; AF043280) obtained from Tococcus salivarius, and its 14 to 512 amino acids were obtained from Streptococcus pneumoniae, It was shown to have 22% identity with ATP binding / permease protein (ComA) (accession number: NP_344591.1; NC_003028) that transports competence factors.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 130 (527 amino acids) is an ABC transporter ATP binding / permease protein (accession number: NP — 2676788.1; NC — 002662) whose 16 to 524 amino acids are obtained from Lactococcus lactis subspecies lactis It has about 23% identity, and its 6-520 amino acids have about 25% identity with the ABC transporter, ATP binding protein (Accession Number: NP_344680.1; NC_003028) obtained from Streptococcus pneumoniae. The ABC transporter ATP binding / transmembrane permease (accession number: NP_357731.1; NC_003098) obtained from Streptococcus pneumoniae About 24% identity with ABC transporter ATP binding / transmembrane protein (accession number: NP_358796.1; NC_003098) with 5% identity and 105-511 amino acids obtained from Streptococcus pneumoniae And its 99-511 amino acids are shown to have 25% identity with the ABC transporter ATP binding protein (Accession Number: NP_490403.1; NC_003276) obtained from Nostock sp. PCC7120. It was.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 132 (529 amino acids) has an ABC transporter ATP binding / permease protein (accession number: NP — 2676788.1; NC — 002662), 10 to 526 amino acids obtained from Lactococcus lactis subspecies lactis Approximately 25% identity, 112-525 amino acids approximately 26% identical to ABC transporter ATP binding / transmembrane permease (accession number: NP — 357731.1; NC — 003098) obtained from Streptococcus pneumoniae ABC transporter, ATP-binding protein (accession number: NP — 344680.1; NC — 00302), from 112 to 525 amino acids obtained from Streptococcus pneumoniae ) And about 107% identity, the 107-518 amino acids of which are homologous to the ABC transporter (TycD) (accession number: pi || T31077) obtained from Brevibacillus brevis. A drug efflux ABC transporter, ATP binding / permease (accession number: NP — 345800.1; NC — 003028), which has about 24% identity with the protein and whose 83-521 amino acids were obtained from Streptococcus pneumoniae It was shown to have 24% identity.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 134 (600 amino acids) is approximately 23% of a protein whose 2-600 amino acids are homologous to ABC transporter (permease) (Accession No: NP_471553.1; NC_003212) obtained from Listeria innocure. About 23% identity with a protein homologous to ABC transporter (permease) (accession number: NP — 465271.1; NC — 003210) whose identity is 1 to 598 amino acids obtained from Listeria monocytogenes 1 to 599 amino acids having about 22% identity with a protein homologous to ABC transporter obtained from Clostridium perfringens (accession number: NP_5616767.1; NC_003366), ~ 564 amino acids have about 22% identity with a protein homologous to ABC transporter obtained from Clostridium perfringens (accession number: NP_561039.1; NC_003366), and 4 ~ 593 amino acids It was shown to have 22% identity with a protein homologous to permease obtained from Clostridium acetobutylicum (accession number: NP_3468681; NC_003030).

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 136 (249 amino acids) has about 58% identity between 1 and 242 amino acids with ABC transporter obtained from Clostridium perfringens (accession number: NP_5617666.1; NC_003366). 3 to 242 amino acids have about 55% identity with a protein homologous to ABC transporter obtained from Clostridium perfringens (accession number: NP_561038.1; NC_003366), and 1 to 242 amino acids thereof Has about 51% identity with a protein homologous to the ABC transporter (ATP binding protein) obtained from Listeria monocytogenes (accession number: NP — 465638.1; NC — 003210). The acid has about 50% identity with a protein homologous to the ABC transporter (ATP binding protein) obtained from Listeria Innocure (accession number: NP_471552.1; NC_003212), of which 3 to 242 amino acids are It was shown to have 54% identity with the ABC transporter, ATP binding protein (Accession Number: NP_346686. 1; NC_003030), obtained from Clostridium acetobutylicum.

  Sequence alignment with BlastP considering gaps showed the following. That is, SEQ ID NO: 138 (423 amino acids) has about 2% 391 amino acid identity with a virtual protein obtained from Streptococcus pyogenes (accession number: NP — 27004.1; NC — 002737), ~ 383 amino acids have about 21% identity with a hypothetical protein obtained from Streptococcus pyogenes (accession number: NP — 608080.1; NC — 003485), 9 to 166 amino acids obtained from Bacillus subtilis, yvbJ An env polyprotein precursor having about 26% identity with a protein (accession number: NP_391268.1; NC_000964), whose 92-281 amino acids are obtained from caprine arthritis-encephalitis virus T It has about 25% identity with protein (accession number: pi || VCLJC6), and its 92-281 amino acids are 24% of envelope glycoprotein (accession number: gb | AAD146661; AF105181). It was shown to have identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 140 (438 amino acids) is approximately 27% identical to its transport aid protein (accession number: gb | AAC95141.1; AF075600) in which 86 to 216 amino acids are obtained from Brochothrix campestris. The amino acid 107-219 has about 26% identity with the bacterosin transport auxiliary protein (Accession No .: NP — 345950.1; NC — 003028) obtained from Streptococcus pneumoniae, the amino acid 107 to 219 Has about 26% identity with the Bta protein obtained from Streptococcus pneumoniae (accession number: gb | AAD566288.1; AF165218), and its 88-201 amino acids are Bacillus anthracis A thioredoxin obtained from Neisseria meningitidis, having 23% identity with a hypothetical protein (Accession number: NP_052783.1; NC_001496) obtained from (Bacillus anthracis) (Accession number: NP_2744384.1; NC_003112) and 32% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 142 (196 amino acids) is approximately 56% identical in that 1-196 amino acids are a protein obtained from Lactobacillus gasseri (accession number: dbj | BAA82351.1; AB029612). 10 to 196 amino acids have about 49% identity with a hypothetical protein (accession number: sp | P29470 | YLA1_LACAC) obtained from a Lactobacillus sp. ABC transporter cofactor obtained from Bacillus casei (accession number: np — 52220.1; nc — 003320) with about 28% identity, whose 90-196 amino acids were obtained from Lactobacillus plantarum, ABC Transporter (PlnH) ABC exporter cofactor (SapE), which has 35% identity with a cofactor (accession number: emb | CAA64190.1; X94434), and whose 41-196 amino acids were obtained from Lactobacillus salmon It was shown to have 30% identity with a protein homologous to (accession number: pi || A56973).

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 144 (720 amino acids) is about 62% identical to ABC transporter (PlnG) (accession number: emb | CAA64189.1; X94434) whose 9-720 amino acids were obtained from Lactobacillus plantarum Approximately 62% of the 6-720 amino acids of which ATP-dependent translocation protein (sppT) (accession number: pi || 57913) is obtained from Lactobacillus sakei 2 to 720 amino acids have about 62% identity with ATP-dependent transfer protein (SapT) (accession number: pi || I56273) obtained from Lactobacillus sakei The 9-720 amino acids were obtained from Lactobacillus casei, ABC transporter (accession number: NP — 604212) which has 62% identity with ABC transporter (accession number: NP — 54229.1; NC — 003320) and whose 25-718 amino acids were obtained from Lactobacillus acidophilus .1; NC_003458) and 57% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 146 (234 amino acids) is a protein in which 13 to 228 amino acids are homologous to ABC transporter ATP-binding protein (accession number: NP_370833.1; NC_002758) obtained from Staphylococcus aureus subspecies aureus And about 11 to 234 amino acids of which are obtained from Streptococcus pyogenes, an ABC transporter (ATP-binding protein) (accession number: NP — 6069694.1; NC — 003485). ABC transporter (ATP binding protein) with 50% identity, 11 to 234 amino acids obtained from Streptococcus pyogenes (accession number: NP_268893.1; ABC transporter ATP-binding protein (accession number: NP_266815.1), which has about 50% identity to a protein homologous to C_002737), 13 to 232 amino acids of which were obtained from Lactococcus lactis subsp. Lactis; NC_002662) with an ABC transporter ATP-binding protein (accession number: NP_268413.1; NC_002662), having 11% to 233 amino acids from Lactococcus lactis subsp. Lactis It was shown to have 53% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 148 (353 amino acids) has about 40% identity between 1 and 352 amino acids with a hypothetical protein obtained from Lactococcus lactis subspecies lactis (accession number: NP — 268412.1; NC — 002662). 1 to 352 amino acids have about 38% identity with the conserved virtual protein obtained from Staphylococcus aureus subspecies Aureus (accession number: NP — 370832.1; NC — 002758), 352 amino acids have approximately 33% identity with the conserved virtual protein obtained from Streptococcus pyogenes (accession number: NP — 268992.1; NC — 002737), 1 to 352 amino acids were obtained from Streptococcus pyogenes Protection ABC transporter permease having 33% identity with the generated virtual protein (accession number: NP — 60693.1; NC — 003485), and 1 to 352 amino acids obtained from Lactococcus lactis subsp. Lactis It was shown to have 34% identity with the protein (accession number: NP — 266816.1; NC — 002662).

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 150 (188 amino acids) has about 47% identity between its 14-85 amino acids and a transcriptional regulator obtained from Lactococcus lactis subspecies lactis (accession number: NP_266817.1; NC_002662). The amino acids 21 to 90 have about 28% identity with the transcriptional regulator of TetR / AcrR family obtained from Aquifex aeolicus (accession number: NP — 213195.1; NC — 000918), Its 14-75 amino acids have about 30% identity with the AcrR family transcriptional regulators (accession number: NP — 348163.1; NC — 003030) obtained from Clostridium acetobutylicum. It has 29% identity with a protein homologous to a transcriptional regulator obtained from P. mycocolor (accession number: emb | CAB930300.1; AL357432), and its 27-88 amino acids are obtained from Clostridium acetobutylicum. In addition, it was shown to have 41% identity with the transcriptional regulatory factors of the TetR / AcrR family (accession number: NP — 347457.1; NC — 003030).

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 152 (236 amino acids) is about 65% identical to ABC transporter ATP binding protein (accession number: NP — 359090.1; NC — 003098) obtained from Streptococcus pneumoniae by 3 to 236 amino acids. 4 to 236 amino acids having about 66% identity with ABC transporter ATP binding protein (Accession Number: NP — 36092.1; NC — 003028) obtained from Streptococcus pneumoniae, and from 4 to 236 amino acids Is approximately 65% identical to a protein homologous to ABC transporter (ATP binding protein) (accession number: NP_607321.1; NC_003485) obtained from Streptococcus pyogenes 4 to 236 amino acids have 65% identity with a protein homologous to ABC transporter (ATP-binding protein) obtained from Streptococcus pyogenes (accession number: NP — 269390.1; NC — 002737), and The 4-236 amino acids have 62% identity with a protein homologous to the ABC transporter, ATP-binding protein (Accession Number: NP_464748.1; NC_003210), obtained from Listeria monocytogenes. It was done.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 154 (846 amino acids) is approximately 41% of the ABC transporter permease protein (accession number: NP — 267260.1; NC — 002662) whose 6 to 846 amino acids are obtained from Lactococcus lactis subspecies lactis. 2-846 amino acids have about 34% identity with a hypothetical protein obtained from Streptococcus pneumoniae (accession number: NP_3590899.1; NC_003098), and 2-846 amino acids are , Approximately 34% identical to a hypothetical protein obtained from Streptococcus pneumoniae (accession number: NP — 346091.1; NC — 003028), 4-846 amino acids obtained from Streptococcus pyogenes A hypothetical protein (accession number: NP — 607320.1; NC — 003485) having 33% identity with the imaginary protein (accession number: NP — 269389.1; NC — 002737) and having 4 to 846 amino acids obtained from Streptococcus pyogenes ) And 33% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 156 (78 amino acids) has about 30% identity in the 12-70 amino acids with a protein obtained from Arabidopsis thaliana (accession number: gb | AAF19707.1; AC008047), 12-70 amino acids have about 30% identity with a protein homologous to the ATP-dependent copper transporter (Accession Number: NP — 176333.1; NM — 105023) obtained from Arabidopsis thaliana, the 1 to 65 amino acids Is approximately 32% identical to a hypothetical protein obtained from Pyrococcus furiosus (accession number: NP — 579673.1; NC — 003413), and its 21 to 55 amino acids are hepatitis TT virus ( Accession number: gb | AA K11712.1; AF345529) and 37% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 158 (379 amino acids) has about 36% identity with its conserved virtual protein (accession number: NP — 470340.1; NC — 003212) whose 32-368 amino acids are obtained from Listeria Inocure , 32 to 353 amino acids have approximately 37% identity with the conserved virtual protein obtained from Listeria monocytogenes (accession number: NP — 464529.1; NC — 003210), and the 87 to 370 amino acids are A hypothetical protein obtained from Lactococcus lactis subspecies lactis having about 36% identity with a protein obtained from Lactococcus lactis (accession number: emb | CAA68042.1; X99710). Protein (Accessory No .: NP — 26788.55.1; NC — 002662), and a protein obtained from Actinosynnema pretiosum subsp. Auranticum (accession) with 32 to 348 amino acids. Number: gb | AAC14002.1; U33059) and 30% identity.

Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 160 (779 amino acids) is approximately 61% of the ABC transporter ATP binding subunit (accession number: gb | AAD09218.1; U73183) whose 1-308 amino acids were obtained from Streptococcus mutans. About 37% of its amino acids 1 to 362 with ABC transporter ATP-binding / permease protein (accession number: NP — 266870.1; NC — 002662) obtained from Lactococcus lactis subspecies lactis About 1 to 295 amino acids having the same identity as a protein homologous to ABC transporter ATP-binding protein (Accession No .: NP_464271.1; NC_003210) obtained from Listeria monocytogenes 9% identity, 1 to 221 amino acids 47% identical to ABC transporter ATP binding protein (accession number: NP_070298.1; NC_000917) obtained from Archaeobus fulgidus And its 1 to 218 amino acids have been shown to have 49% identity with the ABC transporter ATP binding protein (Accession Number: NP_069851.1; NC_000917) obtained from Archeoglobus frugidas. It was.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 162 (38 amino acids) is about 66% identical to mannose-specific phosphotransferase component protein (accession number: NP — 149230.1; NC — 001988) obtained from Clostridium acetobutylicum in 1 to 27 amino acids. About 72% identity with a protein homologous to the PTS mannose-specific factor IIAB (Accession No .: NP — 4636299.1; NC — 003210) obtained from Listeria monocytogenes. About 27% identity with a protein homologous to the PTS mannose-specific factor IIAB (Accession Number: NP — 46948.81; NC — 003212) obtained from Listeria Inocure 1 to 27 amino acids have 66% identity with the PTS protein (Accession Number: NP — 561737.1; NC — 00366) obtained from Clostridium perfringens, and 2 to 27 amino acids are , Obtained from Streptococcus pyogenes, has 65% identity with the mannose-specific phosphotransferase system component IIAB (accession number: NP — 269761.1; NC — 002737).

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 164 (105 amino acids) is a protein whose 1 to 103 amino acids are homologous to PTS mannose-specific factor IIAB (accession number: NP_463629.1; NC_003210) obtained from Listeria monocytogenes. About 59% identity with a protein homologous to PTS mannose-specific factor IIAB (Accession Number: NP — 46948.1; NC — 003212), which has about 60% identity, and whose 1 to 103 amino acids are obtained from Listeria Inocure 1 to 104 amino acids have about 57% identity with PTS proteins (Accession Nos .: NP_561737.1; NC_003366) obtained from Clostridium perfringens. ~ 104 amino acids cross It has 53% identity with mannose-specific phosphotransferase system component IIAB (accession number: NP — 149230.1; NC — 001988) obtained from Tridium acetobutylicum, and its 1 to 96 amino acids are obtained from Streptococcus pyogenes In addition, it was shown to have 54% identity with mannose-specific phosphotransferase system component IIAB (accession number: NP — 607831.1; NC — 003485).

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 166 (269 amino acids) is about 69% of a protein whose 1-269 amino acids are homologous to the PTS mannose-specific factor IIC obtained from Listeria Inocure (accession number: NP — 46949.1; NC — 003212). About 69% of a protein homologous to PTS mannose-specific factor IIC (accession number: NP — 463630.1; NC — 003210) obtained from Listeria monocytogenes. 1 to 269 amino acids having about 67% identity with the PTS mannose-specific IIC component protein (Accession Number: NP — 344821.1; NC — 003028) obtained from Streptococcus pneumoniae, Part 1 269 amino acids have 65% identity with a protein homologous to mannose-specific phosphotransferase system component IIC obtained from Streptococcus pyogenes (accession number: NP — 269762.1; NC — 002737), and its 1 to 269 The amino acid was shown to be 64% identical to the mannose / fructose specific phosphotransferase system component IIC (Accession Number: NP — 149231.1; NC — 001988) obtained from Clostridium acetobutylicum.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 168 (307 amino acids) is about 67 to 67 proteins in which 5 to 307 amino acids are homologous to the PTS mannose-specific factor IID (Accession Number: NP_469490.1; NC_003212) obtained from Listeria innocure. About 67% of the protein having 5% to 307 amino acids and homologous to PTS mannose-specific factor IID (accession number: NP — 463631.1; NC — 003210) obtained from Listeria monocytogenes About 6 to 303 amino acids of which the protein is a mannose-specific phosphotransferase system component IID (accession number: NP — 149232.1; NC — 001988) obtained from Clostridium acetobutylicum Mannose-specific PTS-based component IID (EC 2.7.6.99) with 4% identity, from 4 to 300 amino acids, obtained from Lactococcus lactis subspecies lactis (accession number: NP — 267864. 1; NC_002662) and PTS mannose-specific IID component protein (accession number: NP — 344820.1; NC — 003028), which has 64% identity with 5 to 307 amino acids obtained from Streptococcus pneumoniae It was shown to have 64% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 170 (111 amino acids) has a PTS enzyme II protein (4 to 105 amino acids) having about 51% identity with the protein and 4 to 105 amino acids obtained from Streptococcus pyogenes ( Accession number: NP — 269441.1; NC — 002737), which has about 51% identity, 4 to 110 amino acids obtained from Listeria monocytogenes, cellobiose phosphotransferase enzyme IIB component protein Session No .: NP_466205.1; NC_003210), which has about 54% identity, and whose 4-110 amino acids are obtained from Listeria innocure, is a cellobiose phosphotransferase enzyme IIB component PTS enzyme II (accession number), which has about 54% identity with a protein homologous to the protein (accession number: NP — 472159.1; NC — 003212), 4 to 105 amino acids obtained from Streptococcus pyogenes : NP — 607438.1; NC — 003485), a cellobiose-specific PTS-based IIB component protein (EC2) that has 50% identity with 1 to 109 amino acids obtained from Lactococcus lactis subspecies lactis. 7.1.69) (accession number: NP_2666569.1; NC_002662) was shown to have 50% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 170 (111 amino acids) is approximately 51% of a protein whose 4-105 amino acids are homologous to PTS enzyme II protein (accession number: NP — 269441.1; NC — 002737) obtained from Streptococcus pyogenes. About 54% identical to a protein homologous to the cellobiose phosphotransferase enzyme IIB component protein (accession number: NP_466205.1; NC_003210) obtained from Listeria monocytogenes and having 4 to 110 amino acids The cellobiose phosphotransferase enzyme IIB component (accession number: NP — 472159.1; NC — 003212), whose 4-110 amino acids were obtained from Listeria innocure 50 to 50% identity with a protein homologous to PTS enzyme II (accession number: NP_607438.1; NC_003485) obtained from Streptococcus pyogenes. Cellobiose-specific PTS-based IIB component protein (EC 2.7. 1.69) (Accession number: NP — 26669. 5), with 1 to 109 amino acids obtained from Lactococcus lactis subspecies lactis. 1; NC_002662) and 50% identity.

  Sequence alignment with BlastP (version) considering gaps showed the following. That is, SEQ ID NO: 174 (560 amino acids) has a PTS β-glucoside-specific enzyme II, ABC component protein (accession number: NP — 241162.1; NC — 002570) whose 1 to 551 amino acids were obtained from Bacillus halodurans. The phosphotransferase system (PTS) β-glucoside specific enzyme II, ABC component (accession number: NP — 464265.), whose amino acids 1 to 551 were obtained from Listeria monocytogenes. 1; NC_003210), which has about 39% identity with a protein homologous to NC_003210), 1 to 554 amino acids obtained from Bacillus subtilis, phosphotransferase system (PTS) β-glucoside specific enzyme II, ABC component protein (Accession number: NP_391806.1; NC_000964), PTS β-glucoside-specific IIABC component protein (EIIABC-BGL), which has about 38% identity and 1 to 554 amino acids obtained from Bacillus subtilis ) (Β-glucoside-permease IIABC component) (accession number: sp | P40739 | PTBA_BASU) with 38% identity, 1 to 554 amino acids obtained from Bacillus halodurans PTS β- It was shown to have 37% identity with glucoside specific enzyme II, ABC component protein (accession number: NP — 241461.1; NC — 002570).

  Table 2 shows the top blast results of the sequences indicated by even numbers among SEQ ID NOs: 176 to 364.

Example 2 [PFAM result of amino acid sequence]
The results of topPFAM of the amino acid sequence of the present invention are shown in Table 3.

Example 3 [Glucose Metabolic Gene]
Lactobacillus acidophilus has the ability to utilize a variety of carbohydrates including monosaccharides, disaccharides, and polysaccharides, as shown by its API 50 sugar fermentation pattern. In particular, complex dietary carbohydrates (raffinose, fructooligosaccharides, etc.) that escape digestion in the upper gastrointestinal tract (Gibson et al. (1995) J. Nutr. 125: 1401-1412; Barrangou et al. (2003) Proc. Natl. Acad. Sci. US A 100: 8957-8962). The NCFM genome encodes a wide variety of genes related to carbohydrate utilization, including 20 genes of the phosphoenolpyruvate sugar transferase system (PTS) and the ATP binding cassette (ABC) family of transporters. Five genes are included. Trehalose (ORF 1012) (SEQ ID NOs: 103 and 289), fructose (ORF 1777) (SEQ ID NO: 35), sucrose (ORF 401) (SEQ ID NO: 101), glucose and mannose (ORF 452 (SEQ ID NOs: 1 and 263), ORF 453 (SEQ ID NO: 161), ORF 454 (SEQ ID NO: 163), ORF 455 (SEQ ID NO: 165) and ORF 456 (SEQ ID NO: 167)), melibiose (ORF 1705) (SEQ ID NO: 33), gentiobiose and cellobiose (ORF 1369) (SEQ ID NOs: 17 and 269), Salicin (ORF876) (SEQ ID NO: 169), ORF877 (SEQ ID NO: 3), ORF879 (SEQ ID NO: 171)), Arbutin (ORF884) (SEQ ID NO: 27), and N-acetylglucosamine (ORF14) ) Virtual PTS transporters have been identified for (SEQ ID NO: 21). FOS (ORF 502 (SEQ ID NO: 39 and 273) ORF 504 (SEQ ID NO: 43), ORF 506 (SEQ ID NO: 47)), raffinose (ORF 1439 (SEQ ID NO: 109), ORF 1440 (SEQ ID NO: 111 and 293), ORF 1441 (SEQ ID NO: 113), A virtual ABC transporter for ORF 1442 (SEQ ID NOs: 115 and 295) and maltose (ORF 1854 to ORF 1857) was identified, and a virtual lactose-galactose permease was also identified (ORF 1463) (SEQ ID NO: 175). Most of the gene loci are the same as glycosidases and transcriptional regulatory factors, and local transcriptional control is possible.

  In silico analysis of the genome revealed the presence of genes that represent complete glycolytic pathways. Furthermore, HPr (ORF639 (SEQ ID NO: 177), ptsH), EI (ORF640 (SEQ ID NO: 179), ptsI), CcpA (ORF431 (SEQ ID NO: 181), ccpA), and HPrK / P (ORF676 (SEQ ID NO: 183)) , PtsK), a member of a general carbohydrate utilization regulatory network, has been identified, suggesting an active carbon catabolism suppression network based on sugar availability.

Example 4 [Differentially Expressed Gene]
Cluster analysis using the Ward's hierarchical clustering method, the Volcano plots method, and the counterplot method (Figure 4) was performed on the entire gene expression pattern obtained by growing on 8 different carbohydrates. Eisen et al. (1998) Proc. Natl. Acad. Sci. USA 95: 14863-14868). As a result, 23 to 379 genes, which are 1% to 20% of the genome, respectively, were differentially expressed during the paired processing conditions (p values below Bonferroni correction). All possible treatment comparison methods were examined, and a gene was assumed to be induced when the gene was induced by at least one treatment comparison method. For genes induced by two or more treatment comparison methods, the highest induction level was selected. 342 genes (18% of the genome) showed an induction level of more than 2 times, but only 63 genes (3% of the genome) showed an induction level of more than 4 times that of highly induced genes The number was found to be relatively small. Overall expression levels in most genes were consistent regardless of growth substrate (80% of the genome), but in the selected clusters, the gene and operon were differentially transcribed. Nevertheless, the number of genes specifically induced for each sugar was limited.

  In the presence of glucose, ORF1679 (SEQ ID NO: 133) and ORF1680 (SEQ ID NO: 135) were highly induced compared to other monosaccharides (fructose, galactose) and disaccharides (sucrose, lactose, trehalose). The level of induction when compared to other sugars varies between 3.5 and 6.3 for ORF 1679 (SEQ ID NO: 133) and between 3.7 and 4.7 for ORF 1680 (SEQ ID NO: 135). Was. ORF 1679 (SEQ ID NO: 133) encodes an ABC nucleotide binding protein containing Walker A, Walker B, ABC signature sequences, and commonly found nucleotide binding domain motifs such as the Linton and Higgins motifs. ORF 1680 (SEQ ID NO: 135) encodes an ABC permease with 10 predictive transmembrane domains. Since a protein that binds to a solute is not encoded in the vicinity thereof, it is suggested that such ABC permease may play a role as an exporter rather than as an importer. Several genes and operons including ORF680 (SEQ ID NO: 239) to ORF686 were specifically suppressed by glucose involved in glycogen metabolism. In the presence of a suitable carbon source, such as glucose, energy storage is not required because glycogen is metabolized by the cell to store energy. Other genes such as proteins involved in the uptake of alternative carbohydrate sources and enzymes involved in hydrolysis of such carbohydrates were suppressed in the presence of glucose.

Three genes of the virtual fructose locus, ORF1777 (SEQ ID NO: 35) (FruA, fructose PTS transporter, EIIABC ^Fru ), ORF1778 (SEQ ID NO: 185) (FruK, phosphofructokinase, EC 2.7.1.56) and ORF1779 (SEQ ID NO: 187) (FruR, transcriptional regulatory factor) was differentially expressed. The induction levels of FruA, FruK, and FruR were 3.9, 4.3, and 4.6 or less, respectively. From these results, fructose is transported into the cell via the PTS transporter, converted to fructose-6-phosphate, and this fructose-6-phosphate is a glycolytic intermediate by phosphofructokinase FruK. It is suggested that it is converted to 1,6-bisphosphate.

In the presence of sucrose, three genes at the sucrose locus were differentially expressed. These three genes are ORF399 (SEQ ID NO: 97) (ScrR, transcriptional regulatory factor), ORF400 (SEQ ID NO: 99) (ScrB, sucrose-6-phosphate hydrolase, EC 3.2.1.26), and ORF401. (SEQ ID NO: 101) (ScrA, sucrose PTS transporter EIIBCA ^Suc ). When compared to glucose, induction levels of up to 3.1 with scrR, 2.8 with scrB, and 17.2 with scrA were shown. When compared to monosaccharides and disaccharides, high induction levels of 8.0 to 17.2 were observed, especially with ORF401 (SEQ ID NO: 101). These results indicate that sucrose is transported into the cell via the PTS transporter to become sucrose-6-phosphate and then hydrolyzed to glucose-6-phosphate and fructose by ScrB. .

  Six types of FOS operon genes were differentially expressed. That is, ORF502 (SEQ ID NOs: 39 and 273), ORF503 (SEQ ID NO: 41), ORF504 (SEQ ID NO: 43), ORF506 (SEQ ID NO: 47) (MsmEFFGK ABC transporter), ORF505 (SEQ ID NO: 45) (BfrA, β-fract) Sidase, EC 3.2.1.26) and ORF507 (SEQ ID NO: 49 and 275) (GtfA, sucrose phosphorylase, EC 2.7.1.4) were expressed. The induction levels differed in the range of 15.1 to 40.6 when compared to monosaccharides and disaccharides and differed within the range of 5.5 to 8.9 when compared to raffinose. These results suggest that FOS is transported into the cell via the ABC transporter and then hydrolyzed to fructose and sucrose by fructosidase. Sucrose is then thought to be hydrolyzed to fructose and glucose-1-P by sucrose phosphorylase. FOS induced not only the FOS operon, but also the fructose operon, sucrose PTS transporter, trehalose operon and ABC transporter (ORF 1679 to ORF 1680) (SEQ ID NOs: 133 and 135, respectively).

In the presence of raffinose, six raffinose operon genes were specifically induced. The raffinose loci are ORF1442 (SEQ ID NO: 115 and 295), ORF1441 (SEQ ID NO: 113), ORF1440 (SEQ ID NO: 111 and 293), ORF1439 (SEQ ID NO: 109) (MsmEFFGK ₂ ABC transporter), ORF1438 (SEQ ID NO: 197) (MelA α-galactosidase, EC 3.1.2.22), and ORF 1437 (SEQ ID NO: 195) (GtfA ₂ , sucrose phosphorylase, EC 2.7.1.4). The induction level when compared to all other conditions varied between 15.1 and 45.6. In addition, when compared with other conditions, ORF1433 (SEQ ID NO: 189), 1434 (SEQ ID NO: 191) (dihydroxyacetone kinase, EC 2.7.1.29), and ORF1436 (SEQ ID NO: 193) (glycerol uptake promoting factor) Was induced in the range of 1.9 to 24.7 times.

  In the presence of lactose and galactose, 10 genes distributed at two loci were differentially expressed. The 10 genes are ORF1463 (SEQ ID NO: 175) (LacS permease of the GPH (galactoside-pentose hexuronide) translocator family), ORF1462 (SEQ ID NO: 209) (LacZ, β-galactosidase, EC 3.2.1.23), ORF 1461 (SEQ ID NO: 207), ORF 1460 (SEQ ID NO: 205) (surface protein), ORF 1459 (SEQ ID NO: 203) (GalK, galactokinase, EC 2.7.1.6), ORF 1458 (SEQ ID NO: 201) (GalT, galactose- 1-phosphate uridyl transferase, EC 2.7.7.10), ORF 1457 (SEQ ID NO: 199) (GalM, galactose epimerase, EC 5.1.3.3), ORF 1467 (SEQ ID NO: 211), ORF 1468 (SEQ ID NO: 2) 3) (lacLM, beta-galactosidase, large and small subunits of EC3.2.1.23), and 1469 (SEQ ID NO: 215) (GalE, UDP-glucose epimerase, an EC 5.1.3.2). LacS (SEQ ID NO: 175) is similar to the GPH permease that has already been identified in lactic acid bacteria. LacS (SEQ ID NO: 175) has EIIA at the carboxy terminus but is not a PTS transporter. LacS (SEQ ID NO: 175) has a histidine at position 553. Histidine may be involved in the interaction with HPr as shown by S. salivarius (Lessard et al. (2003) J. Bacteriol. 185: 6764-6772). In the presence of lactose and galactose, galKTM (SEQ ID NOs: 199, 201 and 203) is 3.7 to 17.5 compared to other carbohydrates that do not contain galactose (glucose, fructose, sucrose, trehalose and FOS). 6-fold, lacSZ (SEQ ID NO: 175 and 209) was induced 2.8 to 17.6, and lacL (SEQ ID NO: 213) and galE (SEQ ID NO: 215) were induced 2.7 to 29.5 times. These results suggest that lactose is transported into the cell via the LacS permease of the galactoside-pentose hexuronide translocator family. In the cell, lactose is hydrolyzed to glucose and galactose by LacZ. Thereafter, galactose is phosphorylated by GalK to galactose-1-phosphate, and further converted to UDP-galactose by GalT. UDP-galactose is then epimerized by GalE to UDP-glucose. UDP-glucose appears to be converted to glucose-1-phosphate by ORF1719 (SEQ ID NO: 217) encoding the persistently highly expressed UDP-glucose phosphorylase EC 2.7.7.9. Finally, phosphoglucomutase EC 5.4.2.2 acts on glucose-1-phosphate to produce glucose-6-phosphate, a glycolytic substance.

Three genes at the virtual trehalose locus were also differentially expressed. The trehalose locus is ORF1012 (SEQ ID NOs: 103 and 289) (encoding TreB trehalose PTS transporter EIIABC ^Tre , EC 2.7.6.9), ORF1013 (SEQ ID NO: 105) (TreR, trehalose regulator) and ORF1014 ( SEQ ID NOs: 107 and 291) (TreC, trehalose-6 phosphate hydrolase, EC 3.2.1.93). Induction levels when compared to glucose, sucrose, raffinose, and galactose ranged from 4.3 to 18.6 for treB (SEQ ID NOs: 103 and 289) and 2.3 to 7.3 for treR (SEQ ID NO: 105). And treC (SEQ ID NOs: 107 and 291) were in the range of 2.7 to 18.5. These results suggest that trehalose is transported into cells via the PTS transporter, phosphorylated to trehalose-6 phosphate, and hydrolyzed to glucose and glucose-6 phosphate by TreC.

  Furthermore, the genes showing differential expression include sugar-related genes and energy-related genes, ORF874 (SEQ ID NO: 219) (β-galactosidase, EC3.2.1.86), ORF910 (SEQ ID NO: 221) (L-LDH). , EC 1.1.1.27), ORF 1007 (SEQ ID NO: 223 (pyridoxal kinase 2.7.1.35), ORF 1812 (SEQ ID NO: 225) (α-glucosidase, EC 3.2.1.3), ORF 1632 (SEQ ID NO: 227) (aldehyde dehydrogenase, EC 1.2.1.16), ORF1401 (SEQ ID NO: 229) (NADH peroxidase, EC 1.11.1.1.1), ORF1974 (SEQ ID NO: 231) (pyruvate oxidase, EC1 2.2.3), adhesion genes OR F555, ORF649, ORF1019; aminopeptidase ORF911, ORF1086; amino acid permease, ORF1102 (SEQ ID NO: 233) (membrane protein), ORF1783 (SEQ ID NO: 235) (ABC transporter), and ORF1879 (SEQ ID NO: 237) (pyrimidine kinase, EC 2.7.4.7).

Example 5 [Real-time RT-PCR]
Five genes differentially expressed in the microarray experiment were selected and subjected to a real-time quantitative RT-PCR experiment to examine the effectiveness of the induction level measured by the microarray. These genes were selected based on the broad expression range (−1.52 to +3.87 LSM) and the induction level between sugars (induction level up to 34 times). All the selected genes showed an induction level over 6-fold in at least one case. In addition, the annotations of selected genes were functionally correlated with carbohydrate utilization. The five selected genes were eta-fructosidase (ORF505) (SEQ ID NO: 45), trehalose PTS (ORF1012) (SEQ ID NOs: 103 and 289), glycerol uptake promoting factor (ORF1436) (SEQ ID NO: 193), β-galactosidase ( ORF1467) (SEQ ID NO: 211) and ABC transporter (ORF1679) (SEQ ID NO: 133).

For the five selected genes, the difference in induction level by 6 different treatments was compared, and as a result, 15 induction levels were obtained for each gene. The induction level measured with the microarray was plotted against the induction level measured with Q-PCR to examine the validity of the microarray data. Individual R-square values ranged from 0.642 to 0.883 for each of the genes tested (range from 0.652 to 0.978 when using log ₂ scale data) ). When the data were combined, the overall R-squared value was 0.78 (0.88 when using log ₂ scale data). Correlation analysis was performed with SAS (Cary, NC), and the Spearman test, the Hoeffding test, and the Kendall test showed a correlation in two methods that the P value was less than 0.001. Furthermore, a regression analysis was performed with Excel (Microsoft, CA), and there was a statistically significant (p <1.02 × 10 ⁻²⁵ ) correlation between microarray data and Q-PCR results. Indicated. However, a higher induction level was detected from the measurement by Q-PCR, which is considered to be because the dynamic range of the microarray scanner is smaller than that of the Q-PCR cycler. Similar results have already been reported (Wagner et al. (2003) J. Bacteriol. 185: 2080-2095).

  A comparative analysis of the overall transcriptional profiles for growth in eight carbohydrates identified the basis for carbohydrate transport and catabolism in Lactobacillus acidophilus. Specifically, three different types of carbohydrate transporters (phosphoenolpyruvate, sugar phosphotransferase system (PTS), ATP binding cassette (ABC), and galactoside-pentose hexuronide (GPH) translocator) are differentiated. The types of carbohydrate transporters used by Lactobacillus acidophilus are shown. Transcription profiles suggested that galactosides are transported by GPH translocators, whereas monosaccharides and disaccharides are transported by members of PTS, and polysaccharides are transported by members of the APC family.

From the results of the microarray, fructose is EIIABC ^Fru ORF1777 (SEQ ID NO: 35), sucrose is EIIBCA ^Suc ORF401) (SEQ ID NO: 101), and trehalose is EIIABC ^Tre (ORF1012) (SEQ ID NO: 103 and 289) were shown to be transported respectively. These genes are encoded at the normal PTS locus along with regulators and enzymes that are well characterized in other organisms (FIG. 1). On the other hand, FOS and raffinose are ABC transporters of the MsmEFGK family, ORF 502 (SEQ ID NO: 39 and 273), 503 (SEQ ID NO: 41), 504 (SEQ ID NO: 43), and 505 (SEQ ID NO: 45), and ORF 1437. (SEQ ID NO: 195, ORF 1438 (SEQ ID NO: 197), 1439 (SEQ ID NO: 109), ORF 1440 (SEQ ID NO: 111 and 293), ORF 1441 (SEQ ID NO: 113), and ORF 1442 (SEQ ID NO: 115 and 295), respectively. Microarray results for trehalose and FOS show functional studies that modify the saccharolytic ability of Lactobacillus acidophilus NCFM by knocking out carbohydrate transporters and hydrolases as targets The differential expression of EIIABC ^Tre is consistent with recent studies on Lactobacillus acidophilus that showed that ORF 1012 (SEQ ID NOs: 103 and 289) is involved in trehalose uptake, Similarly, differential expression of the fos operon has been reported on Lactobacillus acidophilus showing that these genes are involved in FOS uptake and catabolism and are induced in the presence of FOS and repressed in the presence of glucose (Barrangou et al. (2003) Proc. Natl. Acad. Sci. USA 100: 8957-8962) In addition, the induction of the raffinose msm locus has been shown to be a streptococcal mutant (Russell et al. (1992)). J. Biol. Chem. 267: 4631-4637) and Streptococcus pneumoniae (Rosenow et al. (1999) Genome Res. 9 : 1189-1197), which is consistent with the reported study.

Many lactic acid bacteria have taken in glucose via a PTS transporter. The EII ^Man PTS transporter has the ability to import both mannose and glucose (Cochu et al. 2003). The Lactobacillus acidophilus mannose PTS system is similar to the Streptococcus thermophilus mannose PTS system with 53-65% identity between proteins and 72-79% similarity. Specifically, EII ^Man is composed of three types of proteins, IIAB ^Man , IIC ^Man , and IID ^Man . IIAB ^Man is ORF452 (SEQ ID NOS: 1 and 263) (manL), and IIC ^Man is ORF455 (SEQ ID NO: 165) (manM) and IID ^Man are encoded by ORF456 (SEQ ID NO: 167) (manN), respectively (FIG. 1). Most of the carbohydrates tested in the present invention specifically induced genes involved in the transport and hydrolysis of the carbohydrate itself, whereas glucose did not. As a result of analyzing mannose PTS, it was revealed that the gene encoding EIIABCD ^Man was continuously highly expressed regardless of the carbohydrate source. Such an expression profile suggests that glucose is a suitable carbohydrate and as already suggested for Lactobacillus plantarum (Kleerebezem et al, (2003) Proc. Natl. Acad. Sci. USA 100: 1990-1995), Lactobacillus acidophilus is also designed so that various carbohydrate sources can be used efficiently.

  Genes that are differentially expressed in the presence of galactose and lactose include the permease (LacS) and the enzymatic mechanism of the Leloir pathway. Members of the LacS subfamily of galactoside-pentose-hexouronide (GPH) translocators include Leuconostoc lactis (Vaughan et al. (1996) Appl. Environ. Microbiol. 62: 1574-1582), Streptococcus Thermophilus (van den Bogaard et al. (2000) J. Bacteriol. 182: 5982-5989), Streptococcus salivarius (Lessard et al. (2003) J. Bacteriol. 185: 6764-6772), and Lactobacillus del A variety of lactic acid bacteria have been described so far, including Brucky (Lapierre et al. (2002) J. Bacteriol. 184: 928-935). LacS has PTSEIIA at the carboxy terminus, but is not a member of the PTS family of transporters. LacS has been reported to have the ability to import both galactose and lactose in selected organisms (Vaughan et al. (1996) Appl. Environ. Microbiol. 62: 1574-1582; van den Bogaard et al (2000) J. Bacteriol. 182: 5982-5989). For the combination of LacS lactose permease with the two subunits of β-galactosidase LacL and LacM, for Lactobacillus plantarum (Kleerebezem et al. 2003) and for Leuconostoc lactis (Vaughan et al. (1996) Appl. Environ. Microbiol. 62: 1574-1582) As already described, Lactobacillus acidophilus has never been reported. Although sustained expression of lacS and lacLM has already been reported (Vaughan et al. (1996) Appl. Environ. Microbiol. 62: 1574-1582), these results indicate that both galactose and lactose uptake and catabolism It shows specific induction of genes involved in. The operon organization for galactoside utilization is variable and unstable in gram-positive bacteria (Lapierre et al. (2002) J. Bacteriol. 184: 928-935; Vaillancourt et al. (2002) J. Bacteriol. 184: 785-793; Boucher et al. (2003) Appl. Environ. Microbiol. 69: 4149-4156; Fortina et al. (2003) Appl. Environ. Microbiol. 69: 3238-3243; Grossiord et al. (2003) J. Bacteriol. 185: 870-878). Lactose-galactose loci are not well conserved even in closely related Lactobacillus species such as Lactobacillus johnsonii, Lactobacillus gasseri, and Lactobacillus acidophilus (Pridmore et al. (2004) Proc. Natl. Acad. Sci. USA 101: 2512-2517).

  The phosphoenolpyruvate or phosphotransferase system is the main sugar transport system of Gram-positive bacteria ((Ajdic et al. (2002) Proc. Natl. Acad. Sci. USA 99: 14434-14439; Warner and Lolkema ( 2003) Microbiol. Mol. Rev. 67: 475-490), this microarray data suggests that the ABC transport system is also important: PTS transporters are involved in monosaccharide and disaccharide uptake However, such carbohydrates are digested in the upper gastrointestinal tract, while oligosaccharides reach the lower intestine and symbiotics compete to obtain other complexes and micronutrients. Below, ABC transporters are more important than PTS in that they have a distinct role in oligosaccharide transport, such as FOS and raffinose. The ability of the host to utilize indigestible nutrients is associated with the competitiveness and persistence of beneficial intestinal flora in the large intestine (Schell et al. (2002) Proc. Natl. Acad. Sci USA 99: 14422-14427).

  Transcription profiles of genes differentially expressed under test conditions suggest that all carbohydrate uptake systems and their respective sugar hydrolases except glucose are specifically induced by their substrates. Furthermore, the genes contained in these inducible loci were repressed in the presence of glucose and the cre sequence was identified in its promoter-operator region. Consensus sequences TGNNWNCGNNWNCA (SEQ ID NO: 365) (Miwa et al. (2000) Nucleic Acids Res. 28: 1206-1210) and TGWAANCGNTNWCA (SEQ ID NO: 366) (Weickert and Chambliss (1990) Proc. Natl. Acad. Sci. USA 87: 6238-6242), a hypothetical catabolic responsive element was searched in the differentially expressed gene and the operon promoter-operator region. Together, these results show regulation and metabolism of carbohydrate uptake at the transcriptional level, suggesting the involvement of an overall regulatory system comparable to carbon catabolism inhibition. Carbon catabolite repression (CCR) regulates carbohydrate transport and transcription of proteins involved in catabolism (Miwa et al. (2000) Nucleic Acids Res. 28: 1206-1210). Catabolic suppression is a widely distributed mechanism in Gram-positive bacteria, and in the cis form, it is mediated by catabolic elements ((Miwa et al. (2000) Nucleic Acids Res. 28: 1206-1210; Wickert and Chambliss (1990) Proc. Natl. Acad. Sci. USA 87: 6238-6242), in the trans type, LacI is involved in the transcriptional repression of genes encoding unwanted carbohydrate degradation components in the presence of suitable substrates. Mediated by a family repressor (Wickert and Chambliss (1990) Proc. Natl. Acad. Sci. USA 87: 6238-6242; Viana et al. (2000) Mol. Microbiol. 36: 570-584; Muscariello et al (2001) Appl. Environ. Microbiol.67: 2903-2907; Warner and Lolkema (2003) Microbiol.Mol. Rev. 67: 475-490) With this regulatory mechanism, cells utilize a variety of carbohydrates. Adjustments can be made, first of all we can focus on suitable energy sources. Based on the major enzymes: HPr (ORF639 (SEQ ID NO: 177), ptsH), EI (ORF640 (SEQ ID NO: 179), ptsI), CcpA (ORF431 (SEQ ID NO: 181), ccpA), and HPrK / P ( ORF676 (SEQ ID NO: 183), ptsK), which are all encoded within the Lactobacillus acidophilus chromosome.

  Inhibition of carbon catabolism has been described for Lactobacillus species (Mahr et al. 2000). PTS is characterized by a phosphate transfer cascade that participates in PEP, EI, HPr, EIIABC and ultimately carries phosphate to carbohydrate substrates (Saier, 2000; Warner and Lolkema, 2003). HPr is an important component of CCR and is regulated through phosphorylation by enzymes I and HPrK / P. When HPr is phosphorylated at histidine at position 15, PTS is activated, and carbohydrates transported via PTS are phosphorylated via EIIABC. On the other hand, when HPr is phosphorylated at serine at position 46, the PTS mechanism does not function (Mijakovic et al. (2002) Proc. Natl. Acad. Sci. USA 99: 13442-13447).

  Although the phosphorylation cascade suggests regulation at the protein level, several references have reported on transcriptional regulation of ccpA and ptsHI. In Streptothermophilus, CcpA is produced by being induced by glucose (van den Bogaart et al. 2000). In some bacteria, carbohydrate sources regulate the transcription level of ptsHI (Luesink et al. 1999). In contrast, in Lactobacillus acidophilus, the expression levels of ccpA, ptsH, ptsI, and ptsK did not change in the presence of different carbohydrates. These results were consistent with regulation via phosphorylation at the protein level. Lactobacillus pentosus ccpA expression level (Mahr et al. (2000) Appl. Environ. Microbiol. 66: 277-283), and Streptococcus thermophilus ptsHI transcription (Cochu et al. (2003) Appl Similar results have been reported for Environ. Microbiol. 69: 5423-5432).

  As a whole, it is possible to reconstruct the carbohydrate transport pathway and the carbohydrate catabolism pathway from the results of the microarray (FIG. 2). Transcription of carbohydrate transporters and hydrolases was specifically induced by their substrates, but the following glycolytic genes were continuously highly expressed. That is, D-lactate dehydrogenase (D-LDH, ORF55 (SEQ ID NO: 241)), phosphoglycerate mutase (PGM, ORF185 (SEQ ID NO: 243)), L-lactate dehydrogenase (L-LDH, ORF271 (SEQ ID NO: 245)) Glyceraldehyde 3-phosphate dehydrogenase (GPDH, ORF698 SEQ ID NO: 247)), phosphoglycerate kinase (PGK, ORF699 (SEQ ID NO: 249)), glucose 6-phosphate isomerase (GPI, ORF752 (SEQ ID NO: 251)) , 2-phosphoglycerate with hydratase (PGDH, ORF889 (SEQ ID NO: 253)), phosphofructokinase (PFK, ORF956 (SEQ ID NO: 255)), pyruvate kinase (PK, ORF957 (SEQ ID NO: 257)), And fructose biphosphate aldolase (FBPA, ORF1599 (SEQ ID NO: 259)) were persistently and highly expressed. Glycerol-3-phosphate ABC transporter (ORF1641 (SEQ ID NO: 261)) is also one of the genes that are continuously highly expressed. In organized carbohydrate uptake, an energy source is obtained from the intestinal environment. It seems that Lactobacillus acidophilus is competing with other symbiotic organisms for nutrients, taking away the opportunity for other bacteria to approach the energy source.

    That is, various carbohydrate uptake systems have been identified and characterized with respect to expression profiles in the presence of various carbohydrates including PTS, ABC and GHP transporters. The uptake and catabolism mechanisms are highly regulated at the transcriptional level, which makes the Lactobacillus acidophilus transcriptome flexible, dynamic, and designed for efficient carbohydrate utilization It was suggested that. Differential gene expression suggested the existence of an extensive carbon catabolite repression regulatory network. Regulatory proteins were persistently highly expressed, suggesting regulation at the protein level rather than at the transcriptional level. From the above, Lactobacillus acidophilus is thought to be able to take in different carbohydrate sources from the complex nutrient environment of the small intestine by efficiently utilizing its metabolic mechanism. In particular, the MsmEFG family of ABC transporters involved in the uptake of FOS and raffinose is important for the ability of Lactobacillus acidophilus to compete for intestinal symbiosis with complex sugars that are not digested by the human host. In conclusion, this information provides new insights into how undigested food compounds affect intestinal bacterial balance. Such studies serve as a model for comparative transcription analysis for bacteria exposed to various growth substrates.

Example 7 [multi-drug transporter]
Microorganisms such as Lactobacillus acidophilus have created various ways to combat the toxicity of antibiotics and other harmful compounds. One is a method involving transporters that promote active excretion of drugs, and drug resistance affects certain microorganisms through the transporters. There are two main classes of multidrug transporters, one of which is a secondary multidrug transporter that uses a transmembrane electrochemical gradient of protons or sodium ions to facilitate drug efflux from cells. The other is an ATP-binding cassette (ABC) type multidrug transporter that uses the free energy of ATP hydrolysis to expel a drug from a cell.

  Secondary multidrug transporters are further classified into several different transport protein families. The major facilitator superfamily (MFS, Pao et al. (1998) Microbiol. Mol. Biol. Rev. 62: 1-34), the small multidrug resistance (SMR) family (Paulsen et al. (1996) Mol. Microbiol .19: 1167-1175), resistance-nodulation-cell division (RND) family (Saier et al. (1994) Mol. Microbiol. 11: 841-847), and multidrug elimination and toxic compound elimination (MATE) It is further classified into the family (Brown et al. (1999) Mol. Microbiol. 31: 394-395). These families are not only associated with multidrug excretion, but their members also include proteins involved in other proton-driven force-dependent transport processes or in other functions.

  MFS membrane transport proteins are proteins involved in the symport, antiport, or uniport of various substrates, including antibiotics (Marger and Saier (1993) Trends Biochem. Sci. 18: 13-20). ). Analysis and alignment of the conserved motifs of resistance-conferring drug efflux proteins revealed that these proteins were divided into two separate clusters, each with 12 or 14 transmembrane segments ( Paulsen and Skurry (1993) Gene124: 1-11). The NCFM genome contains several genes that encode MFS transporters involved in multidrug transport. Among them, ORF552 (SEQ ID NO: 77), ORF566 (SEQ ID NO: 79), ORF567 (SEQ ID NO: 81), ORF1446 (SEQ ID NO: 85), ORF1471 (SEQ ID NO: 87), ORF1621 (SEQ ID NO: 91), ORF1853 (SEQ ID NO: 91) No. 93), ORF 1854 (SEQ ID NO: 321), ORF 164 (SEQ ID NO: 309), ORF 251-253 (SEQ ID NO: 311, 313, 315) and ORF 1062 (SEQ ID NO: 317).

  ABC transporters usually have two hydrophobic membrane domains, each consisting of six hypothetical transmembrane α-helices, and Walker A and Walker B motifs (Walker et al. (1982) EMBO J. 1: 945-951) requires two hydrophilic nucleotide binding domains (NBDs), ie four different domains, and an ABC signature (Hyde et al. (1990) Nature 346: 362-365). These separate domains may be expressed as separate proteins, or in several ways (Faith and Kolter (1993) Microbiol. Rev. 57: 995-1017; Higgens (1992) Annu. Rev. Cell Bio 8: 67-113; Hyde et al. (1990) Nature 346: 362-365). ORF597 (SEQ ID NO: 320) encodes a multidrug ABC transporter in the NCFM genome that has genomic similarity to the Lactococcus lactis ABC multidrug transporter lmrA and Lactobacillus brevis horA.

  All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference, just as each publication or patent application is individually incorporated by reference.

  For clarity of understanding, the present invention has been described in some detail by way of examples and examples, but it will be appreciated that certain changes and modifications may be made to the appended claims.

The target locus. FIG. 4 is a diagram showing the layout of the loci described herein. man: glucose-mannose locus, fru: fructose locus, suc: sucrose locus, fos: FOS locus, raff: raffinose locus, Lac: lactose-galactose locus, tre: trehalose locus, CCR: carbon catabolite Locus. It is a figure which shows carbohydrate utilization in Lactobacillus acidophilus. This scheme shows the carbohydrate transporters and hydrolases predicted from the transcription profile. The protein name and EC classification were identified for each element. The PTS transporter is shown in black. The GPH transporter is shown in light gray.

Claims (20)

An isolated nucleic acid selected from the group consisting of:
a) SEQ ID NOS: 289, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 18 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239 241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 Nucleic acid comprising a nucleotide sequence, or its complementary sequence shown in any combination of 359 and 361 and / or 363;
b) SEQ ID NOS: 289, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 18 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239 241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 Nucleic acid comprising a nucleotide sequence or its complementary sequence having at least 90% sequence identity to the nucleotide sequence shown in any combination of 359 and 361 and / or 363;
c) SEQ ID NOs: 289, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 18 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239 241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 Nucleic acid comprising a fragment of a nucleotide sequence, or its complementary sequence shown in any combination of 359 and 361 and / or 363;
d) SEQ ID NOs: 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 Nucleic acid encoding a polypeptide comprising the amino acid sequence shown in any combination of 8,360,362 and / or 364;
e) SEQ ID NOs: 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 A nucleic acid comprising a nucleotide sequence that encodes a polypeptide having at least 90% amino acid sequence identity to the amino acid sequence shown in any combination of 8, 360, 362 and / or 364; and f) a string with any of ae Nucleic acids that hybridize under mild conditions.   A vector comprising the nucleic acid according to claim 1.   The vector according to claim 2, further comprising a nucleic acid encoding a heteropolypeptide.   A cell comprising the vector according to claim 2. An isolated polypeptide selected from the group consisting of:
a) SEQ ID NOs: 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 Polypeptide comprising an amino acid sequence shown in any combination of 8,360,362 and / or 364;
b) SEQ ID NO: 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 8,360,362 and / or polypeptides comprising a fragment of amino acid sequence shown in any combination of 364;
c) SEQ ID NOs 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 8,360,362 and / or polypeptide comprising 364 with any amino acid sequence shown in the combination of an amino acid sequence having at least 90% sequence identity;
d) SEQ ID NOs: 289, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 18 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239 241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 359, 361 and / or the polypeptide encoded by the nucleotide sequence or its complementary sequence shown in any combination of 363;
e) SEQ ID NOs: 289, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 18 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239 241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 359, 361 and / or a nucleotide sequence shown in any combination of 363 and is encoded by the nucleotide sequence or its complementary sequence having at least 90% sequence identity to a polypeptide that retain activity.   6. The polypeptide of claim 5, further comprising a heteroamino acid sequence.   An antibody that selectively binds to the polypeptide of claim 5. A method for producing a polypeptide, comprising culturing the cell according to claim 4 under conditions where a nucleic acid encoding the polypeptide is expressed, wherein the polypeptide consists of the following a) to e): Method selected from the group:
a) SEQ ID NOs: 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 Polypeptide comprising an amino acid sequence shown in any combination of 8,360,362 and / or 364;
b) SEQ ID NO: 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 8,360,362 and / or polypeptides comprising a fragment of amino acid sequence shown in any combination of 364;
c) SEQ ID NOs 290, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 90, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 3 8,360,362 and / or polypeptide comprising 364 with any amino acid sequence shown in the combination of an amino acid sequence having at least 90% sequence identity;
d) SEQ ID NOs: 289, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 18 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239 241,243,245,247,249,251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341 343, 345, 347, 349, 351, 353, 355, 357 A polypeptide encoded by a nucleotide sequence having at least 90% sequence identity with the nucleotide sequence set forth in any combination of 359, 361 and / or 363, and e) SEQ ID NOs: 289, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 14 151,153,155,157,159,161,163,165,167,169,171,173,175,177,179,181,183,185,187,189,191,193,195,197,199 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249 251,253,255,257,259,261,263,265,267,269,271,273,275,277,279,281,283,285,287,291,293,295,297,299,301 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361 and / or 363 A polypeptide encoded by the nucleotide sequence shown in the combination.   A method for modifying the ability of an organism to transport carbohydrates into and out of a cell, comprising introducing the nucleic acid according to claim 1 into the organism.   A method for modifying the ability of a living organism to accumulate carbohydrates, the method comprising introducing the nucleic acid according to claim 1 into the living organism.   A method for modifying the ability of a living organism to use a carbohydrate as an energy source, the method comprising introducing the nucleic acid according to claim 1 into the living organism.   A method for modifying the flavor of food fermented with microorganisms, the method comprising introducing the nucleic acid according to claim 1 into the microorganism.   A method for modifying the texture of food fermented with microorganisms, the method comprising introducing the nucleic acid according to claim 1 into the microorganism.   A method for modifying the ability of an organism to produce a modified carbohydrate, comprising introducing the nucleic acid of claim 1 into the organism.   A method for altering the ability of an organism to survive under food processing and storage conditions, comprising introducing the nucleic acid of claim 1 into the organism.   A method for modifying the ability of an organism to survive in the digestive tract, comprising introducing the nucleic acid according to claim 1 into the microorganism.   A method for modifying the ability of an organism to produce carbohydrates, comprising introducing the nucleic acid of claim 1 into the organism.   A method of modifying the ability of an organism to transport drugs into and out of a cell, comprising introducing the nucleic acid of claim 1 into the organism.   A plant cell comprising a nucleic acid construct comprising the nucleic acid of claim 1.   A plant produced from the plant cell of claim 19.

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